Chemical Products – James L. Stephenson, Jr., John E. P. Syka, August A. Specht, Thermo Finnigan LLC
Abstract for “Methods to mass spectrometry mixtures of proteins and polypeptides using the proton transfer reaction”
A method involves: (1) extracting a biological sample; (2) repeatedly choosing one from a plurality pre-determined protein/polypeptide analyte compound; (b) injecting a portion into an electrospray-ionization source to generate positive ions, thereby creating a plurality ions; (c) isolating subsets containing respective mass-tocharge (m/z), ranges that correspond to the protonation state for the chosen compound; (d), reacting with ana mass spectrum; (3) identifying the presence of microorganism in the ana sample, using ana based on the analytes; and (es present); and (3) identifying the presence/absence of the sample.
Background for “Methods to mass spectrometry mixtures of proteins and polypeptides using the proton transfer reaction”
Mass spectrometry is gaining popularity as a tool to identify microorganisms. It has a shorter time-to-result and higher accuracy than traditional methods. The most popular mass spectrometry method for microorganism identification is matrix-assisted Laser desorption ionization speed-of-flight mass spectrometry. MALDI-TOF is a method in which cells from an unknown microorganism are mixed together with a suitable UV light absorbing matrix solution, and allowed to dry on a plate. An extract of microbial cell can be used in place of intact cells. After the sample is transferred to the mass spectrometer’s ion source, a laser beam is directed at the sample to desorb and ionize the proteins. Time-dependent mass spectral data are also collected.
“The MALDI-TOF mass spectrum of a microorganism reveals a number peaks from intact proteins, peptides, protein fragments and other molecules that make up the microorganisms’?fingerprint”. This method uses pattern matching to match the peak profiles of unknown microorganisms to a reference database that contains mass spectra of known microorganisms. The higher the confidence level for identification of an organism at the species, genus, or subspecies level, the better the match between the spectrum from the isolated microorganism’s spectrum and the reference database spectrum. The method is based on matching patterns in MALDITOF mass spectrumtra. It does not require that the proteins in the spectrum of an unknown microorganism be identified or otherwise characterized.
MALDI-TOF is fast and inexpensive, but they are limited in their ability to identify pathogens. This includes virulence detection, quantitation, resistance marker detection, strain matching and antibiotic susceptibility testing. A MALDI mass spectrum contains information that reflects the most abundant, ionizable and useful proteins. However, this information is usually limited to ribosomal protein at the experimental conditions. Due to the high conservation of ribosomal protein among prokaryotes it is difficult to differentiate closely related microorganisms using MALDI-TOF. Many ribosomal protein species are closely related and have similar or slightly different amino acids sequences. Single amino acid substitutions are not possible to distinguish with low resolution mass scanners. The detection of other protein markers than ribosomal ones is required to determine the strain, serovar, and antibiotic susceptibility of virulence, as well as other important characteristics. This further limits MALDI-TOF’s ability to perform microbial analysis. To further characterize identified microorganisms, laboratories using MALDITOF must use other methods. MALDI-TOF relies on matching spectral patterns and is therefore not suitable for direct testing, mixed cultures or complex samples containing multiple microorganisms.
“There are many other mass spectrometry methods that can be used to detect microorganisms. Mass spectrometry-based methods for protein sequencing have been described. These include liquid chromatography coupled to tandem mass spectrometry, (LC-MS/MS), and sequence information is obtained using enzyme digests of proteins derived form the microbial sample. This is called “bottom-up” This method, called “bottom-up” proteomics is widely used for protein identification. This method allows for identification of subspecies and strain levels. Chromatographic separation also allows the detection additional proteins. It is useful for characterizing antibiotic resistance markers as well as virulence factors.
“In contrast with?bottom up? proteomics, ?top-down? Top-down proteomics is a method of analyzing protein samples that are not subject to any enzyme, chemical, or other digestion. Top-down analysis allows the study of intact proteins. It permits identification, primary structure determination, and localizations of post-translational modification (PTMs), directly at the protein level. Top-down proteomic analyses typically involve inserting an intact protein into a mass spectrometer’s ionization source, fragmenting the protein and measuring the mass-to charge ratios and the abundances of the fragments. Fragmentation that results is more complicated than a peptide fragmentation may require the use of a mass-spectrometer with high mass accuracy and resolution to interpret the fragmentation pattern. Interpretation generally involves comparing the observed fragmentation pattern with a protein sequence database, which includes both experimental fragmentation results from known samples, or to theoretically predicted fragmentation patterns. For example, Liu et al. (?Top-Down Protein Identification/Characterization of a Priori Unknown Proteins via Ion Trap Collision-Induced Dissociation and Ion/Ion Reactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer?, Anal. Chem. 2009, 81-1441) has described top-down protein identification of unknown modified proteins and their characterization with masses up to?28 kDa.
A top-down analysis has the advantage that a protein can be identified directly rather than being inferred from peptides in bottom-up analyses. Alternative forms of a protein (e.g. Alternative forms of a protein, e.g. post-translational modifications or splice variants may also be identified. Top-down analysis can have a disadvantage over bottom-up because it is more difficult to isolate and purify many proteins. Each protein can be found in an incompletely separated mixture and can yield multiple ion species upon mass spectrometric analyses. Each species corresponds to a different degree of protonation, a different charge state and can lead to multiple isotopic variants. Methods are needed to interpret the highly complex mass spectra.
“Ion-ion reaction have been of great use in biological mass spectrometry for the past decade. This is primarily due to the use of electron transfer disociation (ETD), which allows you to dissociate protein/peptides, determine primary sequence information, and characterize post-translational modification.
“Proton Transfer, another type ion-ion reactions, has been extensively used in biological applications. Experimentally, proton transfer is accomplished by causing multiply-positively-charged protein ions (i.e., protein cations) from a sample to react with singly-charged reagent anions so as to reduce the charge state of an individual protein cation and the number of such charge states of the protein cations. When the reagent anion population is large, these reactions are pseudo-first-order reaction kinetics. The reaction rate is directly proportional the square of the protein cation’s charge (or any other multiply-charged chemical cation), multiplied with the charge on the reagent anon. This same relationship holds true for reactions of opposite polarity. It is the reaction between singly-charged reactive cations and a group of multiply-charged anion derived from a protein sample. This results in a series pseudo-first-order consecutive reaction curves, as determined by the multiply-charged starting protein cation population. The reactions are exothermic and exceed 100 kcal/mol. However, proton transfer is an even electron process that takes place in the presence 1 mtorr background gas (i.e. The proton transfer process does not result in fragmentation of the multiply-charged starting protein cation population. The collision gas removes excess energy at the microsecond scale (108 collisions per seconds), thus preventing fragmentation in the resulting product-ion population.
“Proton Transfer Reactions (PTR) have been successfully used to identify proteins in mixtures. This process of mixture simplification has been used to determine the charge state and molecular masses of high-mass proteins. PTR has also been utilized for simplifying product ion spectra derived from the collisional-activation of multiply-charged precursor protein ions. PTR can reduce the signal derived from multiply charged protein ions but this is more than compensated by the substantial increase in signal-to noise ratio of the resulting PTR productions. PTR is 100% efficient and produces only one series of reaction products. There are no side reactions that need special interpretation or data analysis.
The following documents describe “various aspects of the use of PTR for the analysis of proteins, polypeptides, and peptides”: U.S. Pat. No. 7,749 769 B2 in inventors Hunt et.al., U.S. Patent Preg Grant Publication No. 2012/0156707 B1 in the names inventors Hartmer and al., U.S. Preg Grant Publication No. 2012/0205531 in the name of Zabrouskov; McLuckey, et al.?Ion/IonProton-Transfer Kinetics : Implications For Analysis of Ions Derived From Electrospray of Protein Mixtures? Chem. 1998, 70: 1198-1202; Stephenson and al.,?Ionion Proton Transactions of Bioions Involving Noncovalent Interactions Holomyoglobin? J. Am. Soc. Mass Spectrom. 1998, 8, 637-644. Stephenson et.al.,?Ion/Ion reactions in the Gas Phase? Proton Transfer Reactions Involving Multiply Charged Proteins?, J. Am. Chem. Soc. 1996, 118; McLuckey et.al.,?Ion/Molecule reactions for Improved Effective Mass Resolution In Electrospray Mass Spectrometry?. Anal. Chem. 1995, 67, 2493-2497; Stephenson et al., ?Ion/Ion Proton Transfer Reactions for Protein Mixture Analysis?, Anal. Chem. 1996, 68. 4026-4032. Stephenson et.al.,?Ion/Ion Mixture Analysis: Application of Mixtures Comprised Of 0.5-100 KDa Components?. J. Am. Soc. Mass Spectrom. 1998, 9, 585-596. Stephenson et.al.,?Charge manipulation for improved mass determination of high-mass species and mixture components by electrospray Mass Spectrometry’, J. Mass Spectrom. 1998, 33, 664-672. Stephenson et.al.,?Simplification Of Product Ion Spectra Derived From Multiply Charged Parents Ions via Ion/Ion Chemistry?”, Anal. Chem. 1998, 70: 3533-3544. Scalf et. al., ‘Charge Reduction Electrospray Mass Spectrometry’, Anal. Chem. 2000, 72, 52-60. McLuckey et. al.,?Ion/Ion Chemistry Of High-Mass Multiply Charged Ions? Mass Spectrom., describe various aspects of general ion/ion chemistry. Rev. Rev. No. 7,550,718B2 is the name of McLuckey and al. U.S. Pre Grant Publication No. 62 describes a device for performing PTR and reducing ion charges states in mass spectrometers. 2011/0114835 in the names Chen et. al., U.S. Pre Grant Publication No. 2011/0189788 in the names Brown et. al., U.S. Patent. No. 8,283,626B2 in the names Brown et. al. U.S. Pat. No. 7,518,108 B2 is the name of inventors Frey and al. McLuckey and colleagues have described how to adapt PTR charge reduction techniques for identification and detection of organisms. McLuckey et al. (Electrospray/Ion Trap Mass Spectrometry to the Detection and Identification of Organisms?), Proc. First Joint Services Workshop on Biological Mass Spectrometry. Baltimore, Md. 28-30 Jul. 1997, 127-132).”
“Using a technique called ‘ion parking?, product ions from the PTR can be converted into one or more charge states. To consolidate the PTR productions from variously protonated proteins molecules into particular charge states or states at specific mass-to-charge values, ion parking uses supplementary AC currents. This technique can be used to concentrate the product ion signal into a single or limited number of charge states (and, consequently, into a single or a few respective m/z values) for higher sensitivity detection or further manipulation using collisional-activation, ETD, or other ion manipulation techniques. U.S. Pat. describes a variety of aspects of ion parking. No. 7,064,317B2 in McLuckey’s name; U.S. Patent. No. 7,355,169 B2 under the inventor McLuckey; U.S. Patent. No. 8,334,503B2 in McLuckey’s name; U.S. Patent. No. 8,440,962B2 in the inventor Le Blanc’s name; and in these documents: McLuckey,?Ion Parking during Ion/Ion Responses in Electrodynamic IonTraps?, Anal. Chem. 2002, 74: 336-346. Reid et.al.,?Gas Phase Concentration, Purification and Identification of Whole Proteins From Complex Mixtures?, J. Am. Chem. Soc. 2002, 124; 7353-7362. He et.al.,?Dissociation Of Multiple Protein Ion Charge State Following a Single-Gas-Phase Purification And Concentration Procedure?, Anal. Chem. 2002, 74: 4653-4661. Xia et.al.,?Mutual Storage Mod Ion/Ion Reactions In a Hybrid Linear Ion Ttrap?, J. Am. Soc. Mass. Spectrom. 2005, 16, 71?81; Chrisman and coauthors,?Parallel ion Parking: Improving the Conversion of Parents into First-Generation Electron Transfer Dissociation Products?, Anal. Chem. 2005, 77 (10), 3411-3414, and Chrisman et.al.,?Parallel ion Parking of Protein Mixtures’, Anal. Chem. 2006, 78, 310-316.”
“DISCLOSURE of INVENTION”
“The present disclosure describes an application of ion ion reaction chemistry, in which proton transfers reactions are used to simplify mass spectrometric analysis complex ion populations derived by electrospray Ionization of samples containing mixtures of compounds taken from microorganisms. The inventors have discovered that by subjecting a mass-to-charge-restricted subset of such ions to PTR, the resulting population of product ions comprises a much simpler population of charge states of lower total charge values (where the words ?lower? The inventors discovered that the PTR product ions are a smaller subset of multiply-charged species derived from a complex mixture of charge states than the original precursor ions. This simplified population of charge states has lower total charges. (where?lower? refers to lower or reduced in absolute value). These can easily be resolved and assigned to specific proteins or peptides ions. The PTR productions are a subset of multiplically-charged species that have been derived from a complex mix of charge states. This simplifies mass spectral interpretation. Target analysis can be done using tandem mass spectrometry (MS/MS, MSn) on one protein or another component(s) of a microbial extraction.
“The proton transfer reaction that produces charge-reduced proteins and peptide productions results in mass-to-charge values (m/z), which are higher than the original m/z value. A mixture of different m/z values but different mass and charge can be separated using the micro- or the millisecond timescale. These multiply-charged proteinions with the same m/z values but different mass and charge can be separated using low m/z background ions, which are derived from small molecules or lipids. The multiply-charged proteins are thus separated from the background signal in time, resulting in a separated protein mixture with a high signal-to-noise ratio (s/n). These two factors have led to the discovery that the spectral signatures for the protein/peptide and any other analyte product are significantly different from those of many interferent ions. Multiple stages of PTR reactions are possible to separate protein mixtures using low resolution instruments such as a linear mass spectrometer or ion trap mass-spectrometer. This allows for the separation and isolating of these proteins and other analytes so that MSn analysis can be done. Further, the inventors discovered that simple PTR reactions can be enhanced by using?ion parking. PTR reaction can be used in conjunction with other procedures, allowing an analyst to select or control at least a portion of the product-ion state distribution that results from PTR reaction.
“Alternatively, precise masses or m/z ratios can be described in terms of parts per million (ppm), mass accuracy. Mass spectrometric measurements of proteins and polypeptides require an experimental mass accuracy of 50ppm or higher, more preferably 10ppm, and even more preferably 1ppm. This is because these molecules and their ions often have molecular weights of at minimum 10,000 Da and up to 100,000 Da. As such, accuracy in mass determination or mass analysis is defined as 50 ppm to better, 10 ppm to better, and 1 ppm to better.
“In addition to increasing the signal-to noise ratios for this analysis type, the inventors considered that the reduction in charge on protein ions causes large ions to be refolded in the gas phase. This is described in Zhao and al.,?Effects Of Ion/Ion Propon Transfer Reactions On Conformation of Gas Phase Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cyons Ions?, J. Am. Soc. Mass Spec. 2010, 21, 1208-1217. This results in a smaller configuration that reduces the collisional cross-section of the proteinions and increases their stability against fragmentation due to collision with background gas molecules in the mass analyzer chamber. This effect is especially useful for mass analyzers that use image current detection such as the Fourier-transform ion cyclotron (FT-ICR), or Orbitrap. Thermo Fisher Scientific, Waltham, Mass. sells mass analyzers that use electrostatic trap mass analysisrs. USA). A large amount of energy that is deposited into a protein ion by the PTR process could also be a reason for high mass performance. The PTR process deposits more than 100 kcal/mol of energy. This energy is then effectively dampened with the presence of collision energie. The rapid heating process “boils off?” There are several ways that neutral molecules can be attached to proteins: ion induced dipole, ion induced dipole or dipole-induced dpole interactions. The most important thing is that the reduction in charge state of high-mass proteins can significantly improve the transfer from high pressure ion guides, ion storage, or ion trapping devices where the PTR process commonly occurs, to a lower-pressure region a mass analyzer such as an Orbitrap. mass analyzer. This reduces the kinetic energy of ions, which limits ion scattering, fragmentation and formation of metastable substances. This property, according to the inventors, is particularly important in high-accuracy mass analyses of PTR productions in an accurate mass spectrometer (such as the Orbitrap-type of electrostatic trap Mass analyzer) that detects image currents generated by cyclic Ionic Motion over a longer time period.
The present teachings can be used to identify intact proteins with molecular weights greater than 50 kDa. Researchers have found that the combination of the many advantages noted above can allow for the identification of large peptides or multiple intact proteins from complex mixtures derived naturally from microorganisms. These identifications allow microorganisms to be identified at the subspecies, species and even strain levels. A single or multiple species target protein or polypeptide may be selected to provide an indication based on prior information or knowledge.
The present invention offers an alternative to traditional top-up proteomics methods. It is top-down analysis for intact proteins derived microbial cells using a method that is applicable to nearly all microorganisms, including Gram-positive bacteria and Gram-negative bacteria. The invention allows identification of microorganisms at their genus, species and subspecies, strain pathovar, or serovar levels even when samples contain mixed microorganisms. The methods described herein can also be used to target detection of virulence, antibiotic resistance, susceptibility markers or other characteristics. Because there is no need to chemically or enzymatically digest a sample, the top-down method of the present teachings is quick and easy. Data processing can be done in real time.
Methods that conform to the present teachings can include at least one of the following: microbial cell destruction, solubilization, sample clean up (to remove insoluble components and/or debris and/or concentrate), sample injection or flow injection, quick partial liquid chromatographic separation or standard chromatographic separation and optional mass spectrometry using MS or MS/MS mode. Optionally, the separated range of first-generation PTR products ions may undergo PTR to form second-generation PTR ions. Mass spectrometry using MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/ MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS or any other statistical classification. The mass spectrometry steps should be performed using a high-resolution, high accuracy mass spectrometer. mass analyzer.
“Because the common method uses a limited number of chemical reagents, the methods in the present teachings can be used within an automated system for sample preparation or mass spectrometry. These methods can be automated, from sample preparation to results reporting. The hospital may automatically transfer the results to its electronic medical records system. These records can then be linked directly to patient treatment strategies and billing. This integrated system allows for epidemiological tracking at all levels of the outbreak, including local, regional and global. Multiple systems can be connected to a central computer to integrate data from different instruments before reporting. This is useful for high-throughput laboratories. The system can import data on phenotypic susceptibility, which can be combined with information about identification, virulence and typing generated by the invention.
In a first aspect, it is disclosed a method to identify the presence of a protein/polypeptide, or other biologically relevant compounds within a liquid sample. The method includes: (i) extracting a biological sample; then (b) injecting the extract into an electrospray-ionization source. This generates positive ions that have a variety of mass-to-charge ratio ranges. (c) isolating subsets a number of first-generation ion; (iiiiiiiiiiiiiiiiion species.
In a second embodiment, the electronic control device or processor may also include machine-readable program instructions. This system includes: (1) an electrospray source for sample ions; (2) a mass filtre that receives sample ions from the source; (3) a source for proton transfer reaction (PTR), reagent ions; (4) an Ion trap that receives sample ions from the mass filters; (5) an electronic control unit, or processor, which can be electrically coupled to the source and processor to provide machine-line with a presete ratios. The electronic control unit/processor may also include machine-readable program instructions that can be used to: (1) cause the mass filter to isolate a plurality of sample ions from the source of sample; (2) cause the mass analyzer to generate a mass spectrum of the product ions; (3) a source of proton transfer reaction (PTR) reagent anions from the PTR reagent anion source; (4) enable the mass analyzer to receive and analyze the product ions generated by the reaction of the sample ions.
“The term “real-time spectrum deconvolution” is used. “Spectral deconvolution of mass spectrumral data is done concurrently with an analytical run or mass spectral experiment that generated (or has generated) the data. Mass spectral data obtained by mass analysis of analytes during a gradient treatment may be deconvoluted to identify them. This is done in conjunction with continued collection of mass spectral information of additional analytes eluting at a later retention time. Deconvolution of additional mass spectral information, to identify additional analytes may be done simultaneously with continued collection of mass spectrum data from analytes that have eluted at a third time during the gradient elution. A fast computer may facilitate real-time spectral analysis by using parallel processing or a GPU to do the calculations. Alternately, or in addition, real-time spectrum deconvolution can be made easier by using a computationally efficient algorithm. This could include an algorithm written in assembly language, or making extensive use of in cache look-up tables.
“Real-time” is a more general term. “Real-time” can be used to refer to an activity or event that occurs during a data acquisition process. The data acquisition process itself may include one of more the following individual sub-processes: sample purification (e.g., solid phase extraction, size-exclusion chromatography); sample separation (e.g., chromatography); sample transfer into a mass spectrometer (e.g., infusion or inletting of eluate from a chromatograph); sample ionization in an ion source to as to generate first-generation ions; selection and isolation of ions for further manipulation; causing fragmentation of sample-derived ions or reaction of sample-derived ions with reagent ions so as to generate a first-generation of product ions; optional selection and isolation of product ions; optional further fragmentation of product ions or further reaction of product ions; transfer of ions (first-generation ions or first-generation or subsequent-generation product ions) to a mass analyzer, detection and measurement of ion mass-to-charge ratios by a detector of the mass analyzer; and transfer of data derived from the detection and measurement to a digital processor for storage, mathematical analysis, etc. These events and activities may be performed in “real-time” as defined. They may include: identification or determination, or determination, of the presence, in a specimen, of an analyte; identification or determination, of the existence, in a specimen, of a microorganism; notification to the user about the identification or determination, in a digital processor for storage, mathematical analysis, etc.
“The above-described features and other advantages of the present teachings will be more clearly apparent in the following description and attached claims. Or, you may learn by practicing the invention as described hereinafter.”
“BRIEF DESCRIPTION DES DRAWINGS”
“To clarify the above-mentioned advantages and other features of the present disclosure, a more detailed description will be made by reference to specific embodiments thereof. These are illustrated in the attached drawings. These drawings are not intended to limit the scope of the disclosure. They only illustrate certain embodiments of it. The accompanying drawings will provide additional detail and specificity to the disclosure.
“FIG. “FIG.
“FIG. “FIG.
“FIG. “FIG.
“FIG. 3B is a flow chart of an alternate method according to the present teachings.
“FIG. 3C is a flow chart of an alternative method according to the present teachings.
“FIG. FIG. 3D and FIG. 3D and FIG.
“FIG. “FIG.
“FIG. “FIG.
“FIG. 4B is a spectrum of PTR product-ion mass produced by isolating the ions from the E.coli extract of FIG. 4A is within a 2 Th mass window, centered at m/z=750Th and reacting the isolatedions with PTR-reagent anions.
“FIG. “FIG.
“FIG. 5B is a mass spectrum containing second-generation PTR products ions. It was created by isolating first-generation PTR products ions from FIG. 5A is within a mass window with width 5 Th, centered at 1320 T. The second-generation PTR product ions are then reacting with the first-generation productions with PTR Reagent Anions.
“FIG. “FIG.
“FIG. 6B is the mass spectrum of an isolated PTR product-ion species. It was selected from FIG. 6A, and having an m/z of 833 Th;
“FIG. “FIG. 6B;”
“FIG. 6D is the mass spectrum of an isolated PTR product-ion species, selected from FIG. 6A, and having an m/z of 926 Th;
“FIG. “FIG. 6D;”
“FIG. 6F is the mass spectrum of an isolated PTR product-ion species. It was selected from FIG. 6A, with a m/z ratio 917 Th;
“FIG. “FIG. 6F;”
“FIG. 7A is a schematic depiction of a method, in accordance with the present teachings, of improved-efficiency PTR conversion of ions of a selected analyte to an assemblage of PTR product ions by simultaneous isolation and reaction of multiple m/z ranges of electrospray-produced first-generation precursor ions;”
“FIG. 7B is a schematic diagram of isolation of a first randomly-chosen range of electrospray-produced first-generation precursor ions for PTR reaction, as may be employed in an initial step of a method of improved-efficiency PTR conversion of ions;”
“FIG. 7C shows the recognition of two charge states of PTR productions. They correspond to different analyte molecules. This can be used as an intermediate step in a process of higher-efficiency PTR conversion of Iions.
“FIG. “FIG.
“FIG. 9A is a full-scan mass spectrum of first generation ions, generated from eluate with a retention of 10 minutes. “During a ten minute gradient reverse-phase liquidchromatography separation of an E.coli extract, it took 30 seconds.
“FIG. 9B is a PTR product spectrum ion spectrum created by reacting sulfur Hexafluoride over 10 ms with an isolate population of ions from the sample of FIG. 9A in a 10 Th wide isolation area centered at 750 Th.
“FIG. 10A is a full-scan mass spectrum of first generation ions, generated from eluate with a retention time 42 minutes. ”
“FIG. 10B is a PTR product spectrum ion spectrum created by reacting sulfur Hexafluoride over 10 ms with an isolate population of ions from the sample of FIG. 10A within a 10 Th wide isolation windows centered at 750Th;
“FIG. 11A is a full-scan mass spectrum of first generation ions, generated from eluate with a retention time 18 minutes. ”
“FIG. 11B is a PTR product spectrum ion spectrum created by reaction of PTR-reagent ions with an isolate population of ions from the sample of FIG. 11A in a 10 Th wide isolation area centered at 750 Th.
“FIG. 11C is a full-scan mass spectrum of first generation ions, generated from eluate with a retention time 22 minutes. FIG. 11A; and”
“FIG. 11D is a PTR product spectrum ion spectrum created by reaction of PTR-reagent ions with an isolate population of ions from the sample of FIG. 11C in a 10 Th wide isolation area centered at 750Th.”
“MODES FOR CARRYING THE INVENTION”
“The following description was prepared to allow any person skilled and able to use the invention. It is given in the context of particular applications and their requirements. Many modifications can be made to the described embodiments by those who are skilled in the art. The generic principles may also be applied to other embodiments. The present invention is not limited to the examples and embodiments shown, but should be considered as having a wide range of applications in accordance to the claims. Referring to the attached FIGS will help you see the particular advantages and features of the invention. 1-11, taken together with the following description.
Referring to FIG. Schematically illustrated is a system 100 that extracts proteins from microorganisms. The detection and identification of these proteins are done by FIG. The system 100 comprises a sample handling apparatus 115, which allows for the extraction of proteins from microorganisms. A sample 110 is also accessible via the sample handling tool 115. Sources of reagents and buffers 120 are fluidly connected to the sample handing device 115 using various tubing or other transfer lines. Optionally, the system 100 also includes a first or second sample-purification apparatus 135 (such a solid phase extract cartridge), which is used to clean samples (e.g. removing contaminants, concentrating protein), and an optional chromatography columns 140, which may be used for purifying at least part of a sample 110 using liquid chromatography before mass-spec analysis. One sample-purification device 135 may include an in-line-size exclusion chromatography column. This can be used to remove small molecules and lipids, as well as salts. The fluid communication between the sample 110, the optional second sample-purification device 135, and the optional column 140 is fluid. It also includes a fluid handling pump 130, various reagents, buffers, and other fluids 120, as well as a mass spectrometer150.
The sample handling device 115 can prepare a variety of samples containing microorganisms. It also delivers a soluble protein fraction from these microbes to the mass-spectrometer 150 for analysis. Any type of sample 110 can contain any microorganisms, including isolated colonies from culture plates, blood cultures, saliva, urine and stool.
“The sample handling device (115) may contain one or more of the following: a cell disruption method, a robotic liquid handling mean, a centrifuge and filtration means, an incubator and mixing means, as well as reagents 120 which can be used to isolate soluble proteins fractions from microbes. The mechanical, chemical, enzymatic, and other methods for disrupting bacterial, fungal and mycoplasma cells and viruses can be used. Bead beating, pressure such as French press, and other mechanical methods are all possible. Chemical methods include exposure of chaotropes like urea, Thiourea or Guanidine HCL to lyse microbial cells. Alternately, organic acid/solvents combinations can be used to disrupt cells. To form “holes”, enzymes can be used to create them using lysozyme or lysostaphin, as well as other lytic enzymes. “Leakage of the contents into the surrounding solution occurs in the cell walls of bacterial cells.”
“As shown in FIG. “As illustrated in FIG. The control unit 160 in the illustrated embodiment can also be used as a data processing device to process data from the mass-spectrometer 150, or forward the data to server(s), for storage and processing (the server is not shown). 1). The Control Unit 160 can also measure molecular masses and the charge states of any PTR product ions in real-time for MS/MS, MSn or molecular mass determination. You can use the Control Unit 160 to send the results automatically to healthcare professionals.
“In some embodiments, a system 100 can be used by a physician or a general laboratory technician, who may not have the necessary expertise in sample preparation, LCMS operations, LCMS methods development, or the like. The control unit 160 is designed to protect the data system environment. It provides a user with an easy-to-use interface that allows them to monitor and initiate the assaying of sample 110. The control unit 160 provides a separation between the user’s and the services that control devices, data files, and algorithms to translate data into a user-readable format. The control unit 160 removes the need to know or control hardware used to analyze clinical samples. It also provides an easy interface for sending and receiving information from the mass-spectrometer.
The control unit 160 can be set up to monitor every sample request. It is capable of tracking each request through the system 100 from start to finish. The control unit 160 can be set up to start processing data once sample 110 data has been obtained or acquired by the system 100. This will depend on the type and choice of the user. The control unit 160 can also be set up to process data during acquisition. These results include strain matching information, antibiotic susceptibility testing data, virulence/resistance characterization, microbial identification and virulence characterization. The control unit 160 can also be set up to automatically select the post-processing parameters depending on the type and selection of the user. This reduces the amount of interaction that the user has to make with the system after the assay is selected and initiated for analysis. To reduce the amount of work required to prepare sample assays for analysis, the control unit 160 can be used as a layer between the system 100. To avoid overloading the user with irrelevant information, the control system 160 can be set up to only return the most pertinent data.
“In one embodiment, system 100 may also include a sample detector device (not shown) that can be used in conjunction with or integrated with the sample handler 115. The sample detector device can be used in conjunction with the sample handler 115, or it can function independently. It will perform at least one of these functions: i. Identify samples entering the system; and ii. Identify the assay types of the samples that are entering the system. Select an assay protocol that is based on the type of assay and/or analyte in interest. Direct the sample handling system and/or control system to begin analysis of the analyte in the sample. v. Direct the control system select one or more of the reagents according to the assay protocol for the type and/or interest. vi. Direct the control system, based on the selected assay protocol for the type and/or analyte in interest, to select a liquidchromatography mobile phase condition and allow the liquid chromatography to perform the assay or purify the analyte; vi. Direct the control system, based on the assay protocol chosen for the assay type or analyte in interest, to set a mass-spectrometer setting and cause the mass spectrumrometer to produce mass spectral data related to the selected assay type or analyte in interest. Direct the control system’s analysis of the mass spectral data associated to the selected assay type or analyte to determine the presence or concentration of the analyte.
The sample or processed sample may be cleaned up or purified before analysis by mass spectrometry. This purification or sample clean up may be referred to as a procedure to remove salts and lipids from crude cell extracts, or to enrich one or more of the analytes of particular interest relative to other components of the sample. This could also refer to sample processing in separate laboratories that have biosafety level three facilities for handling mycobacteria and filamentous fungi. This embodiment allows samples to be transferred to the system, and then can be analysed as previously described. One embodiment of this purification or sample clean up may be achieved by a solid phase extractor, in-line exclusion chromatography, and/or the optional column 140.
“In one embodiment, the first or second sample-purification device 135 could include a solid phase extract (SPE) cartridge. The SPE cartridge can be used in conjunction with high resolution/high accuracy mass spectrometer 150. One embodiment of the SPE cartridge could be a tip made from polypropylene with a small volume silica, or another sorbent containing bonded C4, C8 and C18 or other functional group immobilized in it. Cartridge (Thermo Fisher Scientific). Polymeric sorbents and chelating agents can be used in alternative embodiments. You can have a bed volume of 1?L or less, but you may use larger volumes. Because each SPE cartridge can only be used once, there are no carryover issues between samples.
“In one embodiment, the sample-purification device 135 could be an in-line-size-exclusion column that removes small molecules and salts from the sample 110. This method can also be used to separate large molecular-weight proteins. The phases are chosen to be compatible with organic solutions and acids that contain less than 100 percent organic compounds. Phases can be used to accommodate different sizes of protein, ranging in molecular weight between 103 and 108 Da. In order to separate intact proteins from small molecules, flow rates can be adjusted in real-time. Separation flow rates are typically lower than those used to remove small molecules and lipids from the system. To speed up the diffusion of intact proteins, a sample-purification apparatus 135 can be heated. This will significantly reduce run times. You can also divert the flow of mobile phase through sample-purification devices 135 during part of the clean up process to remove impurities and prevent them entering the mass-spectrometer 150.
“In one embodiment, an optional chromatography column 140 could include a column that allows at least partial separation of the protein in the sample. The stationary phase of the chromatography column can be either porous or not-porous silica, agarose particles or a monolithic substance polymerized or other formed within the column. To facilitate separation of proteins, the stationary phase can be coated with a suitable material like C18, C8, or C4 or a suitable derivative. It may also contain a cation exchanger, other material, or a combination of these materials. Chemical bonds may be made to the monoliths or particles within the column. The particle sizes range from 1.5 to 30 millimeters. Pore sizes range from 50 to 300 angstroms. Columns have an average diameter of 50 mm to 2.1mm and a length of about 0.5 to 25 cm. The mobile phase, or eluent, may be a solvent or mixture of solvents. It may also contain salts, acids, or other chemical modifiers. The column separates proteins based on physiochemical properties such as size, net charge and hydrophobicity. One or more chromatographic separation methods can be used, including size exclusion, size exclusion and HILIC, hydrophobic interactions, affinity, normal phase, or reverse-phase.
“Additional methods for purifying samples include liquid chromatography (HPLC, UHPLC), precipitation, solid phase extraction, liquid-liquid extract, dialysis and affinity capture.
There are many methods that use HPLC to clean up samples before mass spectrometry analysis. A skilled person in the art will be able to select the appropriate HPLC columns and instruments for the invention. A medium, which is a packing material, is used to allow for the separation of chemical moieties in time and space. Medium may contain very small particles. These may be bonded to the chemical moieties in order to aid the separation. Hydrophobic bonded surfaces, such as alkyl-bonded surfaces, are suitable. C4, C8, and C18 bonded alkyl group may be used to bond surfaces. Monolithic or other phases may also be used, as per the current state of the art. A chromatographic column has an inlet port to receive a sample, and an outlet port to discharge an effluent containing the fractionated sample. A test sample can be placed at the inlet port and eluted using a solvent/solvent mixture. Then, the column is discharged at its outlet port. Another example is that more than one column can be used simultaneously or as a 2-dimensional (2D) chromatography method. A test sample may then be applied to a column at the inlet, and then eluted using a solvent/solvent mixture onto a second. The second column will then be eluted by a solvent/solvent mixture. There are many solvent options available for the eluting of analytes. You can use a gradient, isocratic, or polytyptic liquid chromatography (i.e. mixed) mode.”
“FIG. “FIG. 1. FIG. 2 shows the mass spectrometer. 2. is a hybrid mass-spectrometer that includes more than one type. The mass spectrometer 150 includes both an Orbitrap and an ion trap mass analyser 216. An electrostatic trap mass analyzer is also included in the analyzer. As different analysis methods, according to the present teachings, use multiple mass analysis data collections, a hybrid mass spectrumrometer system can be used to increase duty cycles by using more than one analyzer simultaneously. Orbitrap? The mass analyzer 212 uses image charge detection. Ions are detected indirectly through detection of an image current that is induced on an electrode by motion of ions in an ion trap.
“In operation of the mass-spectrometer 150a, an electrospay source 201 provides ions from a sample for analysis to an aperture of an skimmer202 at which the ions enter a first vacuum chamber. The ions are then captured by a stacked-ring, ion guide 200 and focussed into a narrow beam. The beam is transferred to the downstream high-vacuum areas of the mass spectrometer by a first ion optical transport component 203. A curved beam guide 206 separates the ion beam from most of the remaining neutral molecules and high-velocity ion groups, including solvated, undesirable ions. The ion clusters and neutral molecules follow a straight line, while the ions of particular interest bend around a ninety degree turn by a dragfield. This causes separation.
“A quadrupole ion mass filter 208 is used with the mass spectrometer150 a in its traditional sense. It acts as a tunable filter to filter out ions within a narrow range of m/z. The filtered ions are delivered to a curved quaddrupole ion trap (C-trap) by a subsequent ion optical transport component 203b. component 210. C-trap210 can transfer ions along the pathway between the quadrupole Mass Filter 208 and ion trap Mass Analyzer 216. C-trap 210 can also temporarily store a population and deliver them to the Orbitrap as a packet or pulse. mass analyzer 212. Control of the transfer of packets is achieved by applying electrical potential differences between C-trap210 and an injection electrodes 211. These electrodes are positioned between C-trap210 and Orbitrap? mass analyzer 212. C-trap curvature is such that the population is spatially concentrated so as to match an Orbitrap’s angular acceptance. Mass analyzer 212”
“Multipole-ion guide214” and optical transfer component203b are used to guide ions between C-trap210 and ion trap mass analyser 216. Multipole ion guide214 allows for temporary storage of ions so that ions generated in one processing step can later be retrieved for processing in another. Multipole ion guides 214 can be used as fragmentation cells. There are several gate electrodes that run along the pathway between C-trap 220 and the ion trap mass analyser 216. These can be controlled so that ions can be transferred either way, depending on the sequence of ion processing steps in each analysis method.
The ion trap mass analyser 216 is a dual pressure linear ion trap. It is a two-dimensional trap that consists of a high-pressure linear trapped cell 217a and a low pressure linear trap cell 227.5b. This plate lens has a small aperture that allows ion transfer between them and also presents a pumping restriction that allows for different pressures to be maintained within the traps. The high-pressure cell 217a is conducive to ion cooling and ion fragmentation through either collision-induced or electron transfer dissociation. The low-pressure cell 217’s environment favors high resolution and mass accuracy analytical scanning. A dual-dynode, ion detector 215 is part of the low-pressure cell.
“Using either an electron transfer dissociation step or a proton transfer reaction in a mass analysis method requires that you can cause controlled ion-ion reactions within a mass spectrumrometer. In order to perform ion-ion reactions, you must be able to generate reagentions and cause the reagentions to mix with the sample ions. FIG. 2 shows the mass spectrometer 150a. FIG. 2 shows two alternative reagent ion sources. A first reagent source 299a is located between the stacked-ring guide 204, and the curved beam guidance 206. A second reagent source 299b is located at the opposite end of instrument, next to the low pressure cell 217b of the linear-ion trap mass analyser 216. A system can only contain one reagent source. Two different sources of reagents ion are shown and discussed in this illustration. The following discussion will be about reagent sources for PTR. However, similar discussions may be applicable to ETD reagents ion source sources.
The stacked ring guide 204 and curved beam guide206 may contain the first possible reagent source 299a. A glow discharge cell is a reagent source 299 a. It consists of a pair electrodes (anode, cathode), that are exposed to a conduit 298 a which delivers the reagent gas (or solid) from a reservoir 297 a. The heater heats the reagent compound. A glow discharge occurs when a high voltage is applied to the electrodes. This ignites the reagent compound between the electrodes. The glow discharge source’s reagent anions are then introduced into the ion optics pathway before the quadrupole Mass Filter 208, where they can be m/z selected. The reagentions can then be stored in the multipole Ion Guide 214 and transferred to the high pressure cell 217b of the dual-pressure line ion trap 216, where they are available for the PTR Reaction. The reaction products can be transferred directly to the Orbitrap 217 a. mass analyzer 212 to do m/z analysis.”
“An alternative source of reagents ions 299 a may be found adjacent to the low pressure linear trap cells 217 b. This chamber may contain an additional high vacuum chamber 292 through which reagents ions can be directed into high pressure cells 217/b through an opening between chamber 292 & the high-pressure cell. Gaseous reagent compounds are supplied from a reagent fluid (or solid) reservoir 297b. This heater volatilizes the compound and then the gas is directed through a conduit 298b that delivers the gas into a partially enclosed ion generation volume 269. The operation involves the transfer of thermionic electrons from an electrically heated filament 244 into an ion-generation volume 296. This is done by applying an electric potential between the filament and an accelerator electrode (not illustrated). The reagent gas is ionized by the energetic electrons. “The reagent ions can then be guided into high pressure cells 217 b using an ion optical transfer device 203 a, under the control of gate electrodes (not illustrated).
The flow diagrams in FIGS. 3A-3F show schematically the examples of methods that conform to the present teachings. 3A-3F. FIG. FIG. The first steps 302, 304, and 306 of method 300 are steps of microorganism destruction (e.g., extraction, solid phase clean-up or size-exclusion/chromatographic separation). The extracted sample can be directly infused into a mass-spectrometer during the next sample introduction step 308 in some cases. You can also prepare samples offline using dialysis or other techniques that are known to the best of our knowledge. In many cases, however, it is useful to use the steps 304 or 306 to purify the sample before mass-spectral analysis.
“When an analysis must take place according to time constraints (e.g. in clinical applications), the time required for the analysis can be reduced by using a SPE step304 or a time-compressed step in chromatography as described in U.S. Pat. No. No. (FPCS) in the step 306 of chromatography as described in international patent publication WO 2013/166169. Generally, FPCS is performed using a crude extract from microbial cells containing a complex mix of organic and inorganic substances (small organic molecules and proteins, as well as their naturally occurring fragments), lipids and nucleic acids, polysaccharides and lipoproteins. The chromatographic column is filled with the analytes and then subjected to chromatography. Instead of allowing each analyte to be eluted separately by a gradient (ideally, one per chromatographic peak), the gradient can be intentionally accelerated so that there are substantially no chromatographicpeaks for example eight minutes or less. Preferably, five minutes or less is preferred to a longer run time to achieve a baseline separation. Many analytes can be co-eluted from the column during the FPCS separation. This is based on their properties and the type chromatography (reverse, HILIC, etc.). used. Other methods, such as the use of mobile phase solvents or modifiers that decrease the retention of compounds on a column, and selection of stationary media that reduce the retention of compounds (including particle size, pores size, etc.) may also be used. Operation of the chromatographic device at a higher flow rate, operation at elevated temperatures, or selections of different chromatographic separation modes (e.g., reversed phase, size exclusion). The FPCS technique produces few, if any, resolved chromatographic peaks over the entire gradient. The time at which the column was eluted from the chromatogram is therefore the most relevant information. Each mass spectrum recorded is a “subset” Co-eluting Analytes are then ionized and separated in the mass analyzer before being detected.”
“In step 308(FIG. “In step 308 (FIG. 3A), the sample goes into a mass-spectrometer. The sample can be supplied as the eluate from an SPE cartridge or chromatography apparatus, or by direct infusion. The sample compounds are then ionized by the mass-spectrometer’s electrospray source (step 308) after being submitted. These electrospray-generated ions are herein referred to as ?first-generation? ions. Optionally, an MS1 scan can be done (step 309) to identify protein-rich areas in the m/z space. Note that the term “scan” is used in this document. (Note that the term “scan” is used in this document to refer to either a mass spectrum as a noun, or to the acquisition of one, when used as verb. The preferred embodiment of the MS1 scan allows for the acquisition of the entire mass range of the mass spectrum instrument. This will allow the user to select, either in data-dependent or independently, the information-rich part of the spectrum to isolate (step 310). In the case of targeted analysis, however, the MS1 scan is not necessary and the execution of the method 300 can proceed directly to step 311. Here, a subset the ions are isolated for further analysis and reaction. If targeted analysis is used, step 310 may allow for the retention of ions within a pre-determined range or multiple ranges of m/z. Ions outside this range or ranges will be discarded. Pre-determined m/z ranges or ranges are selected to correspond to known m/z ratios for targeted analyte protein or peptides, whose presence or quantity can be detected or monitored during the execution of this method.
“Generally speaking, step 310 can be accomplished by injecting the ions from an ion source into an Ion Trap?such as a three-dimensional trap or a curved trap (sometimes called a C-Trap?). A single-segment linear iontrap, multiple-segmented linear iontrap, multipole ionguide, or quadrupole ion filter. Then, resonantly ejecting ions whose m/z values are not within the desired range. This can be done by applying a supplemental AC current across the electrodes or using the appropriate RF/DC ratios to isolate the ion populations of interest. Some embodiments allow the frequency of the additional voltage to be varied so that the ions are released according to their respective m/z ratios. The ions can be detected during ejection to create a mass spectrum. The supplemental AC voltage can be applied to a combination superimposed frequencies so that essentially simultaneous ejections of ions with m/z ratios outside the desired range are possible. In some cases, superimposed frequencies can be combined with multiple segments of missing frequencies (i.e.?notches?). So that ions comprising two or three non-contiguous ranges of m/z ratio are simultaneously isolated in the trap. Each of the non-contiguous ranges of m/z may correspond to a known m/z of a specific targeted analyte protein, or peptide. To isolate specific or targeted mass ranges, the RF/DC voltage ratios applied to a quadrupole weight filter can also be used. To select the best mass isolation windows, a fixed number of RF/DC voltage ratios is used to select specific m/z ranges for the first-generation of ions. This instrument configuration may include a hybrid mass-spectrometer instrument, which could consist of a quadrupole and a C trap, as well as an Orbitrap. A mass analyzer and a high-energy collision cell (HCD), where the isolated population of ions can be stored in the C trap or HCD cells for PTR experiments. The ‘precursor’ is the isolated population or groups of first-generation ions. These ions are subject to subsequent ion-ion reactions and to fragmentation.
In a preferred embodiment, isolation of the precursor population can be done in a first segmented linear trap. The multiply-charged protein population can be moved to another section of the linear iontrap after it has been isolated. This process can be repeated several times to isolate defined ranges precursor ions before the PTR process.
Anions can then be generated by either a rhenium based filament with chemical Ionization or a glow discharge source from a suitable high-electron affinity gaseous reagent. Chemical ionization can also be done using methane, nitrogen, and other gases that are known to exist in the current state of the art. An anion reagent can be either a gas at room temperatures or a liquid with enough vapor pressure to produce excess anions that will drive the PTR reaction under pseudo-first order reactions conditions. The anions are transferred to the trap by using the supplemental AC voltages described above. You can mount the anion source in line with the electrospray source, or on the opposite side of the segmented ion trap. A quadrupole mass filters can also be used to isolate anion and perform subsequent PTR processes in the C trap or HCD cells.
“Multiple-charged anions can be react with singly charged cations in the opposite polarity experiment. They can be derived from proteins and other biomolecules. There are many sources that can produce singly-charged Cations, including chemical, electron and electrospray. These reactions are similar to the previously described reaction kinetics. The most common reagent cations are benzo(f),quinolone and the noble gases, argon, and xenon. Multiply-charged proteins with opposite polarities have been also reacted along with multiply-charged anions derived from nucleic acid using the multiply charged cations of protein.
“In step 314 (FIG. “In step 314 of the method 300 (FIG. In order to determine the mass spectrum, it is possible to detect ions that have been sequentially ejected in the linear or 3D ion trap according their m/z ratios. Alternately, the ions can be directed to another mass analyzer of a mass spectrometer such as a Time-of-Flight mass analyzer (TOF) or an Orbitrap-type electrostatic trap mass analyser to allow for greater accuracy and resolution than what may be possible by sequential scanning the ion trap. The ion trap can be refilled with new precursor ions by sending the product ions to another analyzer while mass analysis is underway. An Orbitrap or FT-ICR mass analyser are examples of the type of mass analyzer that can be used. If the mass analyzer detects image currents generated by cyclic Ion Motion within an iontrap, then the PTR reactions steps may be beneficially reduced collision cross sections of targeted proteins or polypeptide molecules so that they remain stable in the trap for sufficient time to produce high-quality mass spectrumtra. The PTR productions will also have lower kinetic energy as they leave the high pressure C trap region after their transfer to Orbitrap. mass analyzer. The PTR process will result in the complete desolvation of the product ion population, which will enhance the quality and quantity of the mass spectrum.
“The PTR process generates m/z numbers or alternatively, molecular masses from PTR productions. These can be compared to a database that contains individual pathogen standards. The database will contain patient samples and known reference standards. A small number of pathogen identifications can be obtained by matching the m/z or molecular weights to a database that contains individual referenced pathogens. You can limit the subset by using a specific mass accuracy, weighting individual peak intensities, or weighting molecular weight values according to mass in a scoring system. FIG. 402 illustrates this. 3A. 3A. The m/z molecular information will in all likelihood reduce the number of pathogens that can be unambiguously identified by tandem mass spectrometry. This procedure was described in the international (PCT), patent publication WO 2013/166169. To further refine the identification, Bayesian, logistic regression or decision tree-based methods may be used. This m/z (or molecular weight) search can be performed during data acquisition, i.e. while the sample is being analysed. The search can also be done post-acquisition, i.e. after the sample has been analysed. A comparison of five to ten candidate pathogens with a few molecular weights or m/z values (3-10) of proteins will usually be enough to reduce the number. This is illustrated in FIG. 3A.”
“FIG. 3B illustrates schematically a flow diagram for a second exemplary way, method 370, according to the present teachings. FIG. 3B shows the steps 302-314 in the method 370. The steps 302-314 of the method 370 (FIG. 3A), and so the description of these steps will not be repeated. Only the steps that follow the generation of a mass spectrum at step 314, is where the method 370 differs from method 300. This mass spectrum of PTR productions, as described in the method 300, is sufficient to identify and quantify the proteins and polypeptides of your interest. Tandem mass spectrometry, sometimes referred to by MS/MS and MSn, may be required in some cases to resolve any ambiguities in the recognition or quantification of specific proteins or polypeptide molecules. In these cases, PTR reaction products could be considered to consist of a first-generation reaction product which is then fragmented to create a second generation ions. Combining a specific ratio of first-generation reaction products with one or more specific ratios of fragmentions can often allow for identification of specific proteins or polypeptide molecules associated with pathogens. Many times, the same protein that is identified as a pathogen can also be found in similar pathogens. Method 370, specifically tandem mass spectrometry, may be used to identify a single pathogen. It can be used to analyze as many proteins present in a particular PTR fraction or multiple PTR fractions from the same sample.
“Accordingly, steps 318-322 (FIG. 3B) refers to the application of selected reaction monitoring (SRM), or tandem mass spectrometry, as they are applied to the ions created by PTR. If permitted by the mass spectrometry system, some of the PTR productions may already have been stored (immediately following step 312) in an Ion Storage Apparatus of the mass-spectrometer system. The branching step 315 triggers step 317a. In this case, the previously stored ions can be retrieved for further processing. If the previous batch of PTR productions has been exhausted by the mass analysis (step 314) then the alternative step 317b may be required to be executed again to create a new batch.
“In step 318, certain PTR reaction-productions (i.e. the first-generation productions) within a specific m/z range are mass isolated by ejecting any ions whose ratios are not within that range or ranges. In step 320, the isolated ions are then fragmented. The details of the identified charge-state sequence will be used to determine the range or ranges that are chosen. This will be done automatically by the computer. This is known as “data-dependent analysis”. (or ?data-dependent acquisition?, etc.).”
“Data-dependent fragmentation is used in most conventional MS/MS analyses that involve low-mass molecules, usually a few hundred to several thousand Daltons. It involves choosing the highest P number of most abundant precursors. For tandem mass analysis, the data from a previous MS1 data acquisition is used. The number P can be either a constant input or variable input by the user. This data-dependent analysis is not well-suited for multicomponent biopolymer samples. FIG. FIG. 7C shows two charge state distributions. The envelope 905 and envelope 906 denote the respective envelopes. Each envelope corresponds to a different analyte species in this example. Thus, the sets of lines encompassed by envelopes 905 and 906 may be referred to as ?molecular-species-correlative charge-state distributions?. Consider the FIG. 7C is used to represent precursorions. If P=10 the conventional data-dependent fragmentation method would select the ten most solid vertical lines below the envelope 906 for fragmentation. The conventional technique would not choose any of the dotted lines that correspond to envelope 905 The conventional procedure would therefore yield redundant information about the molecule species that corresponds to envelope 906 but not information regarding the molecule species that corresponds to envelope 905.”
“To overcome the shortcomings of conventional data-dependent fragmentation when applied to high-molecular-weight molecules, the inventors have developed the herein-used novel ?top P unique analyte-specific clusters? data-dependent technique so as to replace, for application to high-molecular-weight molecules, the previous ?top P number of the most abundant precursors? logic. Each molecular-species-correlative charge-state distribution is a set of related mass spectral lines (m/z values) which are interpreted, according to the novel ?top P unique analyte-specific clusters? logic to be all generated from one unique molecule. Each molecular-species-correlative charge-state distribution groups together various charge states and isotopic clusters that are indicated to have been generated from a single molecule, prior to ionization. However, the molecular-species-correlative distribution excludes adducts, which are removed prior to data analysis. According to the novel method, fragmentation is performed only on one (or possibly more) selected representatives of a given molecular-species-correlative charge state distribution envelope thereby avoiding the redundancy noted above associated with the conventional data-dependent fragmentation method. The novel?topP unique analyte specific clusters are described. logic, after a representative m/z ratio (or ratios) has been chosen for a first molecular-species-correlative charge-state distribution, any further fragmentation is directed to a representative m/z ratio of the next determined molecular-species-correlative charge-state distribution, and so on.”
“As described previously, isolation in step 318 of method 370 can be achieved by applying an AC voltage to pairs of electrodes. This will cause ions with m/z values that are outside the range of interest to be ejected from traps while ions that are within the range of ranges or ranges are kept within traps. Sometimes, the ion trap that is used for mass isolation might be the same as the one used to perform the full scan mass analysis in step 314.
The supplemental AC voltage applied in mass isolation to an ion trap may include a summation superimposed frequencies so that ions from two or more non-contiguous ranges of m/z are simultaneously isolated. The mass-isolated first generation product ions are then fragmented using a suitable ion fragmentation method, such as collision-induced dissociation. You can fragment the first-generation productions (products ions formed by PTR or original precursor ions) by transfer them in a known manner to a dedicated fragmentation cells. Here, the transferred ions are broken down so that they generate fragment ions. These fragment ions comprise a second generation reaction products. Optionally, some of the fragment productions can be stored for future fragmentation in optional step 321
“In step 322 (FIG. 3B) The fragments created in step 320 are mass-analyzed by a mass analyzer. The mass analyzer must first transfer the second-generation productions to a specific fragmentation cell before performing step 322. An ion trap mass analyser can be used to analyze second-generation productions in step 322. In this case, the mass analyzer used for step 322 could be the same as the one used to perform the full scan mass analysis in step 314. An accurate mass analyzer that can measure mass-to-charge ratios with a precision of 10 ppm?such an FT-ICR or Orbitrap?type electrostatic trap mass analyser?may also be used for step 322.
Summary for “Methods to mass spectrometry mixtures of proteins and polypeptides using the proton transfer reaction”
Mass spectrometry is gaining popularity as a tool to identify microorganisms. It has a shorter time-to-result and higher accuracy than traditional methods. The most popular mass spectrometry method for microorganism identification is matrix-assisted Laser desorption ionization speed-of-flight mass spectrometry. MALDI-TOF is a method in which cells from an unknown microorganism are mixed together with a suitable UV light absorbing matrix solution, and allowed to dry on a plate. An extract of microbial cell can be used in place of intact cells. After the sample is transferred to the mass spectrometer’s ion source, a laser beam is directed at the sample to desorb and ionize the proteins. Time-dependent mass spectral data are also collected.
“The MALDI-TOF mass spectrum of a microorganism reveals a number peaks from intact proteins, peptides, protein fragments and other molecules that make up the microorganisms’?fingerprint”. This method uses pattern matching to match the peak profiles of unknown microorganisms to a reference database that contains mass spectra of known microorganisms. The higher the confidence level for identification of an organism at the species, genus, or subspecies level, the better the match between the spectrum from the isolated microorganism’s spectrum and the reference database spectrum. The method is based on matching patterns in MALDITOF mass spectrumtra. It does not require that the proteins in the spectrum of an unknown microorganism be identified or otherwise characterized.
MALDI-TOF is fast and inexpensive, but they are limited in their ability to identify pathogens. This includes virulence detection, quantitation, resistance marker detection, strain matching and antibiotic susceptibility testing. A MALDI mass spectrum contains information that reflects the most abundant, ionizable and useful proteins. However, this information is usually limited to ribosomal protein at the experimental conditions. Due to the high conservation of ribosomal protein among prokaryotes it is difficult to differentiate closely related microorganisms using MALDI-TOF. Many ribosomal protein species are closely related and have similar or slightly different amino acids sequences. Single amino acid substitutions are not possible to distinguish with low resolution mass scanners. The detection of other protein markers than ribosomal ones is required to determine the strain, serovar, and antibiotic susceptibility of virulence, as well as other important characteristics. This further limits MALDI-TOF’s ability to perform microbial analysis. To further characterize identified microorganisms, laboratories using MALDITOF must use other methods. MALDI-TOF relies on matching spectral patterns and is therefore not suitable for direct testing, mixed cultures or complex samples containing multiple microorganisms.
“There are many other mass spectrometry methods that can be used to detect microorganisms. Mass spectrometry-based methods for protein sequencing have been described. These include liquid chromatography coupled to tandem mass spectrometry, (LC-MS/MS), and sequence information is obtained using enzyme digests of proteins derived form the microbial sample. This is called “bottom-up” This method, called “bottom-up” proteomics is widely used for protein identification. This method allows for identification of subspecies and strain levels. Chromatographic separation also allows the detection additional proteins. It is useful for characterizing antibiotic resistance markers as well as virulence factors.
“In contrast with?bottom up? proteomics, ?top-down? Top-down proteomics is a method of analyzing protein samples that are not subject to any enzyme, chemical, or other digestion. Top-down analysis allows the study of intact proteins. It permits identification, primary structure determination, and localizations of post-translational modification (PTMs), directly at the protein level. Top-down proteomic analyses typically involve inserting an intact protein into a mass spectrometer’s ionization source, fragmenting the protein and measuring the mass-to charge ratios and the abundances of the fragments. Fragmentation that results is more complicated than a peptide fragmentation may require the use of a mass-spectrometer with high mass accuracy and resolution to interpret the fragmentation pattern. Interpretation generally involves comparing the observed fragmentation pattern with a protein sequence database, which includes both experimental fragmentation results from known samples, or to theoretically predicted fragmentation patterns. For example, Liu et al. (?Top-Down Protein Identification/Characterization of a Priori Unknown Proteins via Ion Trap Collision-Induced Dissociation and Ion/Ion Reactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer?, Anal. Chem. 2009, 81-1441) has described top-down protein identification of unknown modified proteins and their characterization with masses up to?28 kDa.
A top-down analysis has the advantage that a protein can be identified directly rather than being inferred from peptides in bottom-up analyses. Alternative forms of a protein (e.g. Alternative forms of a protein, e.g. post-translational modifications or splice variants may also be identified. Top-down analysis can have a disadvantage over bottom-up because it is more difficult to isolate and purify many proteins. Each protein can be found in an incompletely separated mixture and can yield multiple ion species upon mass spectrometric analyses. Each species corresponds to a different degree of protonation, a different charge state and can lead to multiple isotopic variants. Methods are needed to interpret the highly complex mass spectra.
“Ion-ion reaction have been of great use in biological mass spectrometry for the past decade. This is primarily due to the use of electron transfer disociation (ETD), which allows you to dissociate protein/peptides, determine primary sequence information, and characterize post-translational modification.
“Proton Transfer, another type ion-ion reactions, has been extensively used in biological applications. Experimentally, proton transfer is accomplished by causing multiply-positively-charged protein ions (i.e., protein cations) from a sample to react with singly-charged reagent anions so as to reduce the charge state of an individual protein cation and the number of such charge states of the protein cations. When the reagent anion population is large, these reactions are pseudo-first-order reaction kinetics. The reaction rate is directly proportional the square of the protein cation’s charge (or any other multiply-charged chemical cation), multiplied with the charge on the reagent anon. This same relationship holds true for reactions of opposite polarity. It is the reaction between singly-charged reactive cations and a group of multiply-charged anion derived from a protein sample. This results in a series pseudo-first-order consecutive reaction curves, as determined by the multiply-charged starting protein cation population. The reactions are exothermic and exceed 100 kcal/mol. However, proton transfer is an even electron process that takes place in the presence 1 mtorr background gas (i.e. The proton transfer process does not result in fragmentation of the multiply-charged starting protein cation population. The collision gas removes excess energy at the microsecond scale (108 collisions per seconds), thus preventing fragmentation in the resulting product-ion population.
“Proton Transfer Reactions (PTR) have been successfully used to identify proteins in mixtures. This process of mixture simplification has been used to determine the charge state and molecular masses of high-mass proteins. PTR has also been utilized for simplifying product ion spectra derived from the collisional-activation of multiply-charged precursor protein ions. PTR can reduce the signal derived from multiply charged protein ions but this is more than compensated by the substantial increase in signal-to noise ratio of the resulting PTR productions. PTR is 100% efficient and produces only one series of reaction products. There are no side reactions that need special interpretation or data analysis.
The following documents describe “various aspects of the use of PTR for the analysis of proteins, polypeptides, and peptides”: U.S. Pat. No. 7,749 769 B2 in inventors Hunt et.al., U.S. Patent Preg Grant Publication No. 2012/0156707 B1 in the names inventors Hartmer and al., U.S. Preg Grant Publication No. 2012/0205531 in the name of Zabrouskov; McLuckey, et al.?Ion/IonProton-Transfer Kinetics : Implications For Analysis of Ions Derived From Electrospray of Protein Mixtures? Chem. 1998, 70: 1198-1202; Stephenson and al.,?Ionion Proton Transactions of Bioions Involving Noncovalent Interactions Holomyoglobin? J. Am. Soc. Mass Spectrom. 1998, 8, 637-644. Stephenson et.al.,?Ion/Ion reactions in the Gas Phase? Proton Transfer Reactions Involving Multiply Charged Proteins?, J. Am. Chem. Soc. 1996, 118; McLuckey et.al.,?Ion/Molecule reactions for Improved Effective Mass Resolution In Electrospray Mass Spectrometry?. Anal. Chem. 1995, 67, 2493-2497; Stephenson et al., ?Ion/Ion Proton Transfer Reactions for Protein Mixture Analysis?, Anal. Chem. 1996, 68. 4026-4032. Stephenson et.al.,?Ion/Ion Mixture Analysis: Application of Mixtures Comprised Of 0.5-100 KDa Components?. J. Am. Soc. Mass Spectrom. 1998, 9, 585-596. Stephenson et.al.,?Charge manipulation for improved mass determination of high-mass species and mixture components by electrospray Mass Spectrometry’, J. Mass Spectrom. 1998, 33, 664-672. Stephenson et.al.,?Simplification Of Product Ion Spectra Derived From Multiply Charged Parents Ions via Ion/Ion Chemistry?”, Anal. Chem. 1998, 70: 3533-3544. Scalf et. al., ‘Charge Reduction Electrospray Mass Spectrometry’, Anal. Chem. 2000, 72, 52-60. McLuckey et. al.,?Ion/Ion Chemistry Of High-Mass Multiply Charged Ions? Mass Spectrom., describe various aspects of general ion/ion chemistry. Rev. Rev. No. 7,550,718B2 is the name of McLuckey and al. U.S. Pre Grant Publication No. 62 describes a device for performing PTR and reducing ion charges states in mass spectrometers. 2011/0114835 in the names Chen et. al., U.S. Pre Grant Publication No. 2011/0189788 in the names Brown et. al., U.S. Patent. No. 8,283,626B2 in the names Brown et. al. U.S. Pat. No. 7,518,108 B2 is the name of inventors Frey and al. McLuckey and colleagues have described how to adapt PTR charge reduction techniques for identification and detection of organisms. McLuckey et al. (Electrospray/Ion Trap Mass Spectrometry to the Detection and Identification of Organisms?), Proc. First Joint Services Workshop on Biological Mass Spectrometry. Baltimore, Md. 28-30 Jul. 1997, 127-132).”
“Using a technique called ‘ion parking?, product ions from the PTR can be converted into one or more charge states. To consolidate the PTR productions from variously protonated proteins molecules into particular charge states or states at specific mass-to-charge values, ion parking uses supplementary AC currents. This technique can be used to concentrate the product ion signal into a single or limited number of charge states (and, consequently, into a single or a few respective m/z values) for higher sensitivity detection or further manipulation using collisional-activation, ETD, or other ion manipulation techniques. U.S. Pat. describes a variety of aspects of ion parking. No. 7,064,317B2 in McLuckey’s name; U.S. Patent. No. 7,355,169 B2 under the inventor McLuckey; U.S. Patent. No. 8,334,503B2 in McLuckey’s name; U.S. Patent. No. 8,440,962B2 in the inventor Le Blanc’s name; and in these documents: McLuckey,?Ion Parking during Ion/Ion Responses in Electrodynamic IonTraps?, Anal. Chem. 2002, 74: 336-346. Reid et.al.,?Gas Phase Concentration, Purification and Identification of Whole Proteins From Complex Mixtures?, J. Am. Chem. Soc. 2002, 124; 7353-7362. He et.al.,?Dissociation Of Multiple Protein Ion Charge State Following a Single-Gas-Phase Purification And Concentration Procedure?, Anal. Chem. 2002, 74: 4653-4661. Xia et.al.,?Mutual Storage Mod Ion/Ion Reactions In a Hybrid Linear Ion Ttrap?, J. Am. Soc. Mass. Spectrom. 2005, 16, 71?81; Chrisman and coauthors,?Parallel ion Parking: Improving the Conversion of Parents into First-Generation Electron Transfer Dissociation Products?, Anal. Chem. 2005, 77 (10), 3411-3414, and Chrisman et.al.,?Parallel ion Parking of Protein Mixtures’, Anal. Chem. 2006, 78, 310-316.”
“DISCLOSURE of INVENTION”
“The present disclosure describes an application of ion ion reaction chemistry, in which proton transfers reactions are used to simplify mass spectrometric analysis complex ion populations derived by electrospray Ionization of samples containing mixtures of compounds taken from microorganisms. The inventors have discovered that by subjecting a mass-to-charge-restricted subset of such ions to PTR, the resulting population of product ions comprises a much simpler population of charge states of lower total charge values (where the words ?lower? The inventors discovered that the PTR product ions are a smaller subset of multiply-charged species derived from a complex mixture of charge states than the original precursor ions. This simplified population of charge states has lower total charges. (where?lower? refers to lower or reduced in absolute value). These can easily be resolved and assigned to specific proteins or peptides ions. The PTR productions are a subset of multiplically-charged species that have been derived from a complex mix of charge states. This simplifies mass spectral interpretation. Target analysis can be done using tandem mass spectrometry (MS/MS, MSn) on one protein or another component(s) of a microbial extraction.
“The proton transfer reaction that produces charge-reduced proteins and peptide productions results in mass-to-charge values (m/z), which are higher than the original m/z value. A mixture of different m/z values but different mass and charge can be separated using the micro- or the millisecond timescale. These multiply-charged proteinions with the same m/z values but different mass and charge can be separated using low m/z background ions, which are derived from small molecules or lipids. The multiply-charged proteins are thus separated from the background signal in time, resulting in a separated protein mixture with a high signal-to-noise ratio (s/n). These two factors have led to the discovery that the spectral signatures for the protein/peptide and any other analyte product are significantly different from those of many interferent ions. Multiple stages of PTR reactions are possible to separate protein mixtures using low resolution instruments such as a linear mass spectrometer or ion trap mass-spectrometer. This allows for the separation and isolating of these proteins and other analytes so that MSn analysis can be done. Further, the inventors discovered that simple PTR reactions can be enhanced by using?ion parking. PTR reaction can be used in conjunction with other procedures, allowing an analyst to select or control at least a portion of the product-ion state distribution that results from PTR reaction.
“Alternatively, precise masses or m/z ratios can be described in terms of parts per million (ppm), mass accuracy. Mass spectrometric measurements of proteins and polypeptides require an experimental mass accuracy of 50ppm or higher, more preferably 10ppm, and even more preferably 1ppm. This is because these molecules and their ions often have molecular weights of at minimum 10,000 Da and up to 100,000 Da. As such, accuracy in mass determination or mass analysis is defined as 50 ppm to better, 10 ppm to better, and 1 ppm to better.
“In addition to increasing the signal-to noise ratios for this analysis type, the inventors considered that the reduction in charge on protein ions causes large ions to be refolded in the gas phase. This is described in Zhao and al.,?Effects Of Ion/Ion Propon Transfer Reactions On Conformation of Gas Phase Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cytochrome Cyons Ions?, J. Am. Soc. Mass Spec. 2010, 21, 1208-1217. This results in a smaller configuration that reduces the collisional cross-section of the proteinions and increases their stability against fragmentation due to collision with background gas molecules in the mass analyzer chamber. This effect is especially useful for mass analyzers that use image current detection such as the Fourier-transform ion cyclotron (FT-ICR), or Orbitrap. Thermo Fisher Scientific, Waltham, Mass. sells mass analyzers that use electrostatic trap mass analysisrs. USA). A large amount of energy that is deposited into a protein ion by the PTR process could also be a reason for high mass performance. The PTR process deposits more than 100 kcal/mol of energy. This energy is then effectively dampened with the presence of collision energie. The rapid heating process “boils off?” There are several ways that neutral molecules can be attached to proteins: ion induced dipole, ion induced dipole or dipole-induced dpole interactions. The most important thing is that the reduction in charge state of high-mass proteins can significantly improve the transfer from high pressure ion guides, ion storage, or ion trapping devices where the PTR process commonly occurs, to a lower-pressure region a mass analyzer such as an Orbitrap. mass analyzer. This reduces the kinetic energy of ions, which limits ion scattering, fragmentation and formation of metastable substances. This property, according to the inventors, is particularly important in high-accuracy mass analyses of PTR productions in an accurate mass spectrometer (such as the Orbitrap-type of electrostatic trap Mass analyzer) that detects image currents generated by cyclic Ionic Motion over a longer time period.
The present teachings can be used to identify intact proteins with molecular weights greater than 50 kDa. Researchers have found that the combination of the many advantages noted above can allow for the identification of large peptides or multiple intact proteins from complex mixtures derived naturally from microorganisms. These identifications allow microorganisms to be identified at the subspecies, species and even strain levels. A single or multiple species target protein or polypeptide may be selected to provide an indication based on prior information or knowledge.
The present invention offers an alternative to traditional top-up proteomics methods. It is top-down analysis for intact proteins derived microbial cells using a method that is applicable to nearly all microorganisms, including Gram-positive bacteria and Gram-negative bacteria. The invention allows identification of microorganisms at their genus, species and subspecies, strain pathovar, or serovar levels even when samples contain mixed microorganisms. The methods described herein can also be used to target detection of virulence, antibiotic resistance, susceptibility markers or other characteristics. Because there is no need to chemically or enzymatically digest a sample, the top-down method of the present teachings is quick and easy. Data processing can be done in real time.
Methods that conform to the present teachings can include at least one of the following: microbial cell destruction, solubilization, sample clean up (to remove insoluble components and/or debris and/or concentrate), sample injection or flow injection, quick partial liquid chromatographic separation or standard chromatographic separation and optional mass spectrometry using MS or MS/MS mode. Optionally, the separated range of first-generation PTR products ions may undergo PTR to form second-generation PTR ions. Mass spectrometry using MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/ MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS/MS or any other statistical classification. The mass spectrometry steps should be performed using a high-resolution, high accuracy mass spectrometer. mass analyzer.
“Because the common method uses a limited number of chemical reagents, the methods in the present teachings can be used within an automated system for sample preparation or mass spectrometry. These methods can be automated, from sample preparation to results reporting. The hospital may automatically transfer the results to its electronic medical records system. These records can then be linked directly to patient treatment strategies and billing. This integrated system allows for epidemiological tracking at all levels of the outbreak, including local, regional and global. Multiple systems can be connected to a central computer to integrate data from different instruments before reporting. This is useful for high-throughput laboratories. The system can import data on phenotypic susceptibility, which can be combined with information about identification, virulence and typing generated by the invention.
In a first aspect, it is disclosed a method to identify the presence of a protein/polypeptide, or other biologically relevant compounds within a liquid sample. The method includes: (i) extracting a biological sample; then (b) injecting the extract into an electrospray-ionization source. This generates positive ions that have a variety of mass-to-charge ratio ranges. (c) isolating subsets a number of first-generation ion; (iiiiiiiiiiiiiiiiion species.
In a second embodiment, the electronic control device or processor may also include machine-readable program instructions. This system includes: (1) an electrospray source for sample ions; (2) a mass filtre that receives sample ions from the source; (3) a source for proton transfer reaction (PTR), reagent ions; (4) an Ion trap that receives sample ions from the mass filters; (5) an electronic control unit, or processor, which can be electrically coupled to the source and processor to provide machine-line with a presete ratios. The electronic control unit/processor may also include machine-readable program instructions that can be used to: (1) cause the mass filter to isolate a plurality of sample ions from the source of sample; (2) cause the mass analyzer to generate a mass spectrum of the product ions; (3) a source of proton transfer reaction (PTR) reagent anions from the PTR reagent anion source; (4) enable the mass analyzer to receive and analyze the product ions generated by the reaction of the sample ions.
“The term “real-time spectrum deconvolution” is used. “Spectral deconvolution of mass spectrumral data is done concurrently with an analytical run or mass spectral experiment that generated (or has generated) the data. Mass spectral data obtained by mass analysis of analytes during a gradient treatment may be deconvoluted to identify them. This is done in conjunction with continued collection of mass spectral information of additional analytes eluting at a later retention time. Deconvolution of additional mass spectral information, to identify additional analytes may be done simultaneously with continued collection of mass spectrum data from analytes that have eluted at a third time during the gradient elution. A fast computer may facilitate real-time spectral analysis by using parallel processing or a GPU to do the calculations. Alternately, or in addition, real-time spectrum deconvolution can be made easier by using a computationally efficient algorithm. This could include an algorithm written in assembly language, or making extensive use of in cache look-up tables.
“Real-time” is a more general term. “Real-time” can be used to refer to an activity or event that occurs during a data acquisition process. The data acquisition process itself may include one of more the following individual sub-processes: sample purification (e.g., solid phase extraction, size-exclusion chromatography); sample separation (e.g., chromatography); sample transfer into a mass spectrometer (e.g., infusion or inletting of eluate from a chromatograph); sample ionization in an ion source to as to generate first-generation ions; selection and isolation of ions for further manipulation; causing fragmentation of sample-derived ions or reaction of sample-derived ions with reagent ions so as to generate a first-generation of product ions; optional selection and isolation of product ions; optional further fragmentation of product ions or further reaction of product ions; transfer of ions (first-generation ions or first-generation or subsequent-generation product ions) to a mass analyzer, detection and measurement of ion mass-to-charge ratios by a detector of the mass analyzer; and transfer of data derived from the detection and measurement to a digital processor for storage, mathematical analysis, etc. These events and activities may be performed in “real-time” as defined. They may include: identification or determination, or determination, of the presence, in a specimen, of an analyte; identification or determination, of the existence, in a specimen, of a microorganism; notification to the user about the identification or determination, in a digital processor for storage, mathematical analysis, etc.
“The above-described features and other advantages of the present teachings will be more clearly apparent in the following description and attached claims. Or, you may learn by practicing the invention as described hereinafter.”
“BRIEF DESCRIPTION DES DRAWINGS”
“To clarify the above-mentioned advantages and other features of the present disclosure, a more detailed description will be made by reference to specific embodiments thereof. These are illustrated in the attached drawings. These drawings are not intended to limit the scope of the disclosure. They only illustrate certain embodiments of it. The accompanying drawings will provide additional detail and specificity to the disclosure.
“FIG. “FIG.
“FIG. “FIG.
“FIG. “FIG.
“FIG. 3B is a flow chart of an alternate method according to the present teachings.
“FIG. 3C is a flow chart of an alternative method according to the present teachings.
“FIG. FIG. 3D and FIG. 3D and FIG.
“FIG. “FIG.
“FIG. “FIG.
“FIG. 4B is a spectrum of PTR product-ion mass produced by isolating the ions from the E.coli extract of FIG. 4A is within a 2 Th mass window, centered at m/z=750Th and reacting the isolatedions with PTR-reagent anions.
“FIG. “FIG.
“FIG. 5B is a mass spectrum containing second-generation PTR products ions. It was created by isolating first-generation PTR products ions from FIG. 5A is within a mass window with width 5 Th, centered at 1320 T. The second-generation PTR product ions are then reacting with the first-generation productions with PTR Reagent Anions.
“FIG. “FIG.
“FIG. 6B is the mass spectrum of an isolated PTR product-ion species. It was selected from FIG. 6A, and having an m/z of 833 Th;
“FIG. “FIG. 6B;”
“FIG. 6D is the mass spectrum of an isolated PTR product-ion species, selected from FIG. 6A, and having an m/z of 926 Th;
“FIG. “FIG. 6D;”
“FIG. 6F is the mass spectrum of an isolated PTR product-ion species. It was selected from FIG. 6A, with a m/z ratio 917 Th;
“FIG. “FIG. 6F;”
“FIG. 7A is a schematic depiction of a method, in accordance with the present teachings, of improved-efficiency PTR conversion of ions of a selected analyte to an assemblage of PTR product ions by simultaneous isolation and reaction of multiple m/z ranges of electrospray-produced first-generation precursor ions;”
“FIG. 7B is a schematic diagram of isolation of a first randomly-chosen range of electrospray-produced first-generation precursor ions for PTR reaction, as may be employed in an initial step of a method of improved-efficiency PTR conversion of ions;”
“FIG. 7C shows the recognition of two charge states of PTR productions. They correspond to different analyte molecules. This can be used as an intermediate step in a process of higher-efficiency PTR conversion of Iions.
“FIG. “FIG.
“FIG. 9A is a full-scan mass spectrum of first generation ions, generated from eluate with a retention of 10 minutes. “During a ten minute gradient reverse-phase liquidchromatography separation of an E.coli extract, it took 30 seconds.
“FIG. 9B is a PTR product spectrum ion spectrum created by reacting sulfur Hexafluoride over 10 ms with an isolate population of ions from the sample of FIG. 9A in a 10 Th wide isolation area centered at 750 Th.
“FIG. 10A is a full-scan mass spectrum of first generation ions, generated from eluate with a retention time 42 minutes. ”
“FIG. 10B is a PTR product spectrum ion spectrum created by reacting sulfur Hexafluoride over 10 ms with an isolate population of ions from the sample of FIG. 10A within a 10 Th wide isolation windows centered at 750Th;
“FIG. 11A is a full-scan mass spectrum of first generation ions, generated from eluate with a retention time 18 minutes. ”
“FIG. 11B is a PTR product spectrum ion spectrum created by reaction of PTR-reagent ions with an isolate population of ions from the sample of FIG. 11A in a 10 Th wide isolation area centered at 750 Th.
“FIG. 11C is a full-scan mass spectrum of first generation ions, generated from eluate with a retention time 22 minutes. FIG. 11A; and”
“FIG. 11D is a PTR product spectrum ion spectrum created by reaction of PTR-reagent ions with an isolate population of ions from the sample of FIG. 11C in a 10 Th wide isolation area centered at 750Th.”
“MODES FOR CARRYING THE INVENTION”
“The following description was prepared to allow any person skilled and able to use the invention. It is given in the context of particular applications and their requirements. Many modifications can be made to the described embodiments by those who are skilled in the art. The generic principles may also be applied to other embodiments. The present invention is not limited to the examples and embodiments shown, but should be considered as having a wide range of applications in accordance to the claims. Referring to the attached FIGS will help you see the particular advantages and features of the invention. 1-11, taken together with the following description.
Referring to FIG. Schematically illustrated is a system 100 that extracts proteins from microorganisms. The detection and identification of these proteins are done by FIG. The system 100 comprises a sample handling apparatus 115, which allows for the extraction of proteins from microorganisms. A sample 110 is also accessible via the sample handling tool 115. Sources of reagents and buffers 120 are fluidly connected to the sample handing device 115 using various tubing or other transfer lines. Optionally, the system 100 also includes a first or second sample-purification apparatus 135 (such a solid phase extract cartridge), which is used to clean samples (e.g. removing contaminants, concentrating protein), and an optional chromatography columns 140, which may be used for purifying at least part of a sample 110 using liquid chromatography before mass-spec analysis. One sample-purification device 135 may include an in-line-size exclusion chromatography column. This can be used to remove small molecules and lipids, as well as salts. The fluid communication between the sample 110, the optional second sample-purification device 135, and the optional column 140 is fluid. It also includes a fluid handling pump 130, various reagents, buffers, and other fluids 120, as well as a mass spectrometer150.
The sample handling device 115 can prepare a variety of samples containing microorganisms. It also delivers a soluble protein fraction from these microbes to the mass-spectrometer 150 for analysis. Any type of sample 110 can contain any microorganisms, including isolated colonies from culture plates, blood cultures, saliva, urine and stool.
“The sample handling device (115) may contain one or more of the following: a cell disruption method, a robotic liquid handling mean, a centrifuge and filtration means, an incubator and mixing means, as well as reagents 120 which can be used to isolate soluble proteins fractions from microbes. The mechanical, chemical, enzymatic, and other methods for disrupting bacterial, fungal and mycoplasma cells and viruses can be used. Bead beating, pressure such as French press, and other mechanical methods are all possible. Chemical methods include exposure of chaotropes like urea, Thiourea or Guanidine HCL to lyse microbial cells. Alternately, organic acid/solvents combinations can be used to disrupt cells. To form “holes”, enzymes can be used to create them using lysozyme or lysostaphin, as well as other lytic enzymes. “Leakage of the contents into the surrounding solution occurs in the cell walls of bacterial cells.”
“As shown in FIG. “As illustrated in FIG. The control unit 160 in the illustrated embodiment can also be used as a data processing device to process data from the mass-spectrometer 150, or forward the data to server(s), for storage and processing (the server is not shown). 1). The Control Unit 160 can also measure molecular masses and the charge states of any PTR product ions in real-time for MS/MS, MSn or molecular mass determination. You can use the Control Unit 160 to send the results automatically to healthcare professionals.
“In some embodiments, a system 100 can be used by a physician or a general laboratory technician, who may not have the necessary expertise in sample preparation, LCMS operations, LCMS methods development, or the like. The control unit 160 is designed to protect the data system environment. It provides a user with an easy-to-use interface that allows them to monitor and initiate the assaying of sample 110. The control unit 160 provides a separation between the user’s and the services that control devices, data files, and algorithms to translate data into a user-readable format. The control unit 160 removes the need to know or control hardware used to analyze clinical samples. It also provides an easy interface for sending and receiving information from the mass-spectrometer.
The control unit 160 can be set up to monitor every sample request. It is capable of tracking each request through the system 100 from start to finish. The control unit 160 can be set up to start processing data once sample 110 data has been obtained or acquired by the system 100. This will depend on the type and choice of the user. The control unit 160 can also be set up to process data during acquisition. These results include strain matching information, antibiotic susceptibility testing data, virulence/resistance characterization, microbial identification and virulence characterization. The control unit 160 can also be set up to automatically select the post-processing parameters depending on the type and selection of the user. This reduces the amount of interaction that the user has to make with the system after the assay is selected and initiated for analysis. To reduce the amount of work required to prepare sample assays for analysis, the control unit 160 can be used as a layer between the system 100. To avoid overloading the user with irrelevant information, the control system 160 can be set up to only return the most pertinent data.
“In one embodiment, system 100 may also include a sample detector device (not shown) that can be used in conjunction with or integrated with the sample handler 115. The sample detector device can be used in conjunction with the sample handler 115, or it can function independently. It will perform at least one of these functions: i. Identify samples entering the system; and ii. Identify the assay types of the samples that are entering the system. Select an assay protocol that is based on the type of assay and/or analyte in interest. Direct the sample handling system and/or control system to begin analysis of the analyte in the sample. v. Direct the control system select one or more of the reagents according to the assay protocol for the type and/or interest. vi. Direct the control system, based on the selected assay protocol for the type and/or analyte in interest, to select a liquidchromatography mobile phase condition and allow the liquid chromatography to perform the assay or purify the analyte; vi. Direct the control system, based on the assay protocol chosen for the assay type or analyte in interest, to set a mass-spectrometer setting and cause the mass spectrumrometer to produce mass spectral data related to the selected assay type or analyte in interest. Direct the control system’s analysis of the mass spectral data associated to the selected assay type or analyte to determine the presence or concentration of the analyte.
The sample or processed sample may be cleaned up or purified before analysis by mass spectrometry. This purification or sample clean up may be referred to as a procedure to remove salts and lipids from crude cell extracts, or to enrich one or more of the analytes of particular interest relative to other components of the sample. This could also refer to sample processing in separate laboratories that have biosafety level three facilities for handling mycobacteria and filamentous fungi. This embodiment allows samples to be transferred to the system, and then can be analysed as previously described. One embodiment of this purification or sample clean up may be achieved by a solid phase extractor, in-line exclusion chromatography, and/or the optional column 140.
“In one embodiment, the first or second sample-purification device 135 could include a solid phase extract (SPE) cartridge. The SPE cartridge can be used in conjunction with high resolution/high accuracy mass spectrometer 150. One embodiment of the SPE cartridge could be a tip made from polypropylene with a small volume silica, or another sorbent containing bonded C4, C8 and C18 or other functional group immobilized in it. Cartridge (Thermo Fisher Scientific). Polymeric sorbents and chelating agents can be used in alternative embodiments. You can have a bed volume of 1?L or less, but you may use larger volumes. Because each SPE cartridge can only be used once, there are no carryover issues between samples.
“In one embodiment, the sample-purification device 135 could be an in-line-size-exclusion column that removes small molecules and salts from the sample 110. This method can also be used to separate large molecular-weight proteins. The phases are chosen to be compatible with organic solutions and acids that contain less than 100 percent organic compounds. Phases can be used to accommodate different sizes of protein, ranging in molecular weight between 103 and 108 Da. In order to separate intact proteins from small molecules, flow rates can be adjusted in real-time. Separation flow rates are typically lower than those used to remove small molecules and lipids from the system. To speed up the diffusion of intact proteins, a sample-purification apparatus 135 can be heated. This will significantly reduce run times. You can also divert the flow of mobile phase through sample-purification devices 135 during part of the clean up process to remove impurities and prevent them entering the mass-spectrometer 150.
“In one embodiment, an optional chromatography column 140 could include a column that allows at least partial separation of the protein in the sample. The stationary phase of the chromatography column can be either porous or not-porous silica, agarose particles or a monolithic substance polymerized or other formed within the column. To facilitate separation of proteins, the stationary phase can be coated with a suitable material like C18, C8, or C4 or a suitable derivative. It may also contain a cation exchanger, other material, or a combination of these materials. Chemical bonds may be made to the monoliths or particles within the column. The particle sizes range from 1.5 to 30 millimeters. Pore sizes range from 50 to 300 angstroms. Columns have an average diameter of 50 mm to 2.1mm and a length of about 0.5 to 25 cm. The mobile phase, or eluent, may be a solvent or mixture of solvents. It may also contain salts, acids, or other chemical modifiers. The column separates proteins based on physiochemical properties such as size, net charge and hydrophobicity. One or more chromatographic separation methods can be used, including size exclusion, size exclusion and HILIC, hydrophobic interactions, affinity, normal phase, or reverse-phase.
“Additional methods for purifying samples include liquid chromatography (HPLC, UHPLC), precipitation, solid phase extraction, liquid-liquid extract, dialysis and affinity capture.
There are many methods that use HPLC to clean up samples before mass spectrometry analysis. A skilled person in the art will be able to select the appropriate HPLC columns and instruments for the invention. A medium, which is a packing material, is used to allow for the separation of chemical moieties in time and space. Medium may contain very small particles. These may be bonded to the chemical moieties in order to aid the separation. Hydrophobic bonded surfaces, such as alkyl-bonded surfaces, are suitable. C4, C8, and C18 bonded alkyl group may be used to bond surfaces. Monolithic or other phases may also be used, as per the current state of the art. A chromatographic column has an inlet port to receive a sample, and an outlet port to discharge an effluent containing the fractionated sample. A test sample can be placed at the inlet port and eluted using a solvent/solvent mixture. Then, the column is discharged at its outlet port. Another example is that more than one column can be used simultaneously or as a 2-dimensional (2D) chromatography method. A test sample may then be applied to a column at the inlet, and then eluted using a solvent/solvent mixture onto a second. The second column will then be eluted by a solvent/solvent mixture. There are many solvent options available for the eluting of analytes. You can use a gradient, isocratic, or polytyptic liquid chromatography (i.e. mixed) mode.”
“FIG. “FIG. 1. FIG. 2 shows the mass spectrometer. 2. is a hybrid mass-spectrometer that includes more than one type. The mass spectrometer 150 includes both an Orbitrap and an ion trap mass analyser 216. An electrostatic trap mass analyzer is also included in the analyzer. As different analysis methods, according to the present teachings, use multiple mass analysis data collections, a hybrid mass spectrumrometer system can be used to increase duty cycles by using more than one analyzer simultaneously. Orbitrap? The mass analyzer 212 uses image charge detection. Ions are detected indirectly through detection of an image current that is induced on an electrode by motion of ions in an ion trap.
“In operation of the mass-spectrometer 150a, an electrospay source 201 provides ions from a sample for analysis to an aperture of an skimmer202 at which the ions enter a first vacuum chamber. The ions are then captured by a stacked-ring, ion guide 200 and focussed into a narrow beam. The beam is transferred to the downstream high-vacuum areas of the mass spectrometer by a first ion optical transport component 203. A curved beam guide 206 separates the ion beam from most of the remaining neutral molecules and high-velocity ion groups, including solvated, undesirable ions. The ion clusters and neutral molecules follow a straight line, while the ions of particular interest bend around a ninety degree turn by a dragfield. This causes separation.
“A quadrupole ion mass filter 208 is used with the mass spectrometer150 a in its traditional sense. It acts as a tunable filter to filter out ions within a narrow range of m/z. The filtered ions are delivered to a curved quaddrupole ion trap (C-trap) by a subsequent ion optical transport component 203b. component 210. C-trap210 can transfer ions along the pathway between the quadrupole Mass Filter 208 and ion trap Mass Analyzer 216. C-trap 210 can also temporarily store a population and deliver them to the Orbitrap as a packet or pulse. mass analyzer 212. Control of the transfer of packets is achieved by applying electrical potential differences between C-trap210 and an injection electrodes 211. These electrodes are positioned between C-trap210 and Orbitrap? mass analyzer 212. C-trap curvature is such that the population is spatially concentrated so as to match an Orbitrap’s angular acceptance. Mass analyzer 212”
“Multipole-ion guide214” and optical transfer component203b are used to guide ions between C-trap210 and ion trap mass analyser 216. Multipole ion guide214 allows for temporary storage of ions so that ions generated in one processing step can later be retrieved for processing in another. Multipole ion guides 214 can be used as fragmentation cells. There are several gate electrodes that run along the pathway between C-trap 220 and the ion trap mass analyser 216. These can be controlled so that ions can be transferred either way, depending on the sequence of ion processing steps in each analysis method.
The ion trap mass analyser 216 is a dual pressure linear ion trap. It is a two-dimensional trap that consists of a high-pressure linear trapped cell 217a and a low pressure linear trap cell 227.5b. This plate lens has a small aperture that allows ion transfer between them and also presents a pumping restriction that allows for different pressures to be maintained within the traps. The high-pressure cell 217a is conducive to ion cooling and ion fragmentation through either collision-induced or electron transfer dissociation. The low-pressure cell 217’s environment favors high resolution and mass accuracy analytical scanning. A dual-dynode, ion detector 215 is part of the low-pressure cell.
“Using either an electron transfer dissociation step or a proton transfer reaction in a mass analysis method requires that you can cause controlled ion-ion reactions within a mass spectrumrometer. In order to perform ion-ion reactions, you must be able to generate reagentions and cause the reagentions to mix with the sample ions. FIG. 2 shows the mass spectrometer 150a. FIG. 2 shows two alternative reagent ion sources. A first reagent source 299a is located between the stacked-ring guide 204, and the curved beam guidance 206. A second reagent source 299b is located at the opposite end of instrument, next to the low pressure cell 217b of the linear-ion trap mass analyser 216. A system can only contain one reagent source. Two different sources of reagents ion are shown and discussed in this illustration. The following discussion will be about reagent sources for PTR. However, similar discussions may be applicable to ETD reagents ion source sources.
The stacked ring guide 204 and curved beam guide206 may contain the first possible reagent source 299a. A glow discharge cell is a reagent source 299 a. It consists of a pair electrodes (anode, cathode), that are exposed to a conduit 298 a which delivers the reagent gas (or solid) from a reservoir 297 a. The heater heats the reagent compound. A glow discharge occurs when a high voltage is applied to the electrodes. This ignites the reagent compound between the electrodes. The glow discharge source’s reagent anions are then introduced into the ion optics pathway before the quadrupole Mass Filter 208, where they can be m/z selected. The reagentions can then be stored in the multipole Ion Guide 214 and transferred to the high pressure cell 217b of the dual-pressure line ion trap 216, where they are available for the PTR Reaction. The reaction products can be transferred directly to the Orbitrap 217 a. mass analyzer 212 to do m/z analysis.”
“An alternative source of reagents ions 299 a may be found adjacent to the low pressure linear trap cells 217 b. This chamber may contain an additional high vacuum chamber 292 through which reagents ions can be directed into high pressure cells 217/b through an opening between chamber 292 & the high-pressure cell. Gaseous reagent compounds are supplied from a reagent fluid (or solid) reservoir 297b. This heater volatilizes the compound and then the gas is directed through a conduit 298b that delivers the gas into a partially enclosed ion generation volume 269. The operation involves the transfer of thermionic electrons from an electrically heated filament 244 into an ion-generation volume 296. This is done by applying an electric potential between the filament and an accelerator electrode (not illustrated). The reagent gas is ionized by the energetic electrons. “The reagent ions can then be guided into high pressure cells 217 b using an ion optical transfer device 203 a, under the control of gate electrodes (not illustrated).
The flow diagrams in FIGS. 3A-3F show schematically the examples of methods that conform to the present teachings. 3A-3F. FIG. FIG. The first steps 302, 304, and 306 of method 300 are steps of microorganism destruction (e.g., extraction, solid phase clean-up or size-exclusion/chromatographic separation). The extracted sample can be directly infused into a mass-spectrometer during the next sample introduction step 308 in some cases. You can also prepare samples offline using dialysis or other techniques that are known to the best of our knowledge. In many cases, however, it is useful to use the steps 304 or 306 to purify the sample before mass-spectral analysis.
“When an analysis must take place according to time constraints (e.g. in clinical applications), the time required for the analysis can be reduced by using a SPE step304 or a time-compressed step in chromatography as described in U.S. Pat. No. No. (FPCS) in the step 306 of chromatography as described in international patent publication WO 2013/166169. Generally, FPCS is performed using a crude extract from microbial cells containing a complex mix of organic and inorganic substances (small organic molecules and proteins, as well as their naturally occurring fragments), lipids and nucleic acids, polysaccharides and lipoproteins. The chromatographic column is filled with the analytes and then subjected to chromatography. Instead of allowing each analyte to be eluted separately by a gradient (ideally, one per chromatographic peak), the gradient can be intentionally accelerated so that there are substantially no chromatographicpeaks for example eight minutes or less. Preferably, five minutes or less is preferred to a longer run time to achieve a baseline separation. Many analytes can be co-eluted from the column during the FPCS separation. This is based on their properties and the type chromatography (reverse, HILIC, etc.). used. Other methods, such as the use of mobile phase solvents or modifiers that decrease the retention of compounds on a column, and selection of stationary media that reduce the retention of compounds (including particle size, pores size, etc.) may also be used. Operation of the chromatographic device at a higher flow rate, operation at elevated temperatures, or selections of different chromatographic separation modes (e.g., reversed phase, size exclusion). The FPCS technique produces few, if any, resolved chromatographic peaks over the entire gradient. The time at which the column was eluted from the chromatogram is therefore the most relevant information. Each mass spectrum recorded is a “subset” Co-eluting Analytes are then ionized and separated in the mass analyzer before being detected.”
“In step 308(FIG. “In step 308 (FIG. 3A), the sample goes into a mass-spectrometer. The sample can be supplied as the eluate from an SPE cartridge or chromatography apparatus, or by direct infusion. The sample compounds are then ionized by the mass-spectrometer’s electrospray source (step 308) after being submitted. These electrospray-generated ions are herein referred to as ?first-generation? ions. Optionally, an MS1 scan can be done (step 309) to identify protein-rich areas in the m/z space. Note that the term “scan” is used in this document. (Note that the term “scan” is used in this document to refer to either a mass spectrum as a noun, or to the acquisition of one, when used as verb. The preferred embodiment of the MS1 scan allows for the acquisition of the entire mass range of the mass spectrum instrument. This will allow the user to select, either in data-dependent or independently, the information-rich part of the spectrum to isolate (step 310). In the case of targeted analysis, however, the MS1 scan is not necessary and the execution of the method 300 can proceed directly to step 311. Here, a subset the ions are isolated for further analysis and reaction. If targeted analysis is used, step 310 may allow for the retention of ions within a pre-determined range or multiple ranges of m/z. Ions outside this range or ranges will be discarded. Pre-determined m/z ranges or ranges are selected to correspond to known m/z ratios for targeted analyte protein or peptides, whose presence or quantity can be detected or monitored during the execution of this method.
“Generally speaking, step 310 can be accomplished by injecting the ions from an ion source into an Ion Trap?such as a three-dimensional trap or a curved trap (sometimes called a C-Trap?). A single-segment linear iontrap, multiple-segmented linear iontrap, multipole ionguide, or quadrupole ion filter. Then, resonantly ejecting ions whose m/z values are not within the desired range. This can be done by applying a supplemental AC current across the electrodes or using the appropriate RF/DC ratios to isolate the ion populations of interest. Some embodiments allow the frequency of the additional voltage to be varied so that the ions are released according to their respective m/z ratios. The ions can be detected during ejection to create a mass spectrum. The supplemental AC voltage can be applied to a combination superimposed frequencies so that essentially simultaneous ejections of ions with m/z ratios outside the desired range are possible. In some cases, superimposed frequencies can be combined with multiple segments of missing frequencies (i.e.?notches?). So that ions comprising two or three non-contiguous ranges of m/z ratio are simultaneously isolated in the trap. Each of the non-contiguous ranges of m/z may correspond to a known m/z of a specific targeted analyte protein, or peptide. To isolate specific or targeted mass ranges, the RF/DC voltage ratios applied to a quadrupole weight filter can also be used. To select the best mass isolation windows, a fixed number of RF/DC voltage ratios is used to select specific m/z ranges for the first-generation of ions. This instrument configuration may include a hybrid mass-spectrometer instrument, which could consist of a quadrupole and a C trap, as well as an Orbitrap. A mass analyzer and a high-energy collision cell (HCD), where the isolated population of ions can be stored in the C trap or HCD cells for PTR experiments. The ‘precursor’ is the isolated population or groups of first-generation ions. These ions are subject to subsequent ion-ion reactions and to fragmentation.
In a preferred embodiment, isolation of the precursor population can be done in a first segmented linear trap. The multiply-charged protein population can be moved to another section of the linear iontrap after it has been isolated. This process can be repeated several times to isolate defined ranges precursor ions before the PTR process.
Anions can then be generated by either a rhenium based filament with chemical Ionization or a glow discharge source from a suitable high-electron affinity gaseous reagent. Chemical ionization can also be done using methane, nitrogen, and other gases that are known to exist in the current state of the art. An anion reagent can be either a gas at room temperatures or a liquid with enough vapor pressure to produce excess anions that will drive the PTR reaction under pseudo-first order reactions conditions. The anions are transferred to the trap by using the supplemental AC voltages described above. You can mount the anion source in line with the electrospray source, or on the opposite side of the segmented ion trap. A quadrupole mass filters can also be used to isolate anion and perform subsequent PTR processes in the C trap or HCD cells.
“Multiple-charged anions can be react with singly charged cations in the opposite polarity experiment. They can be derived from proteins and other biomolecules. There are many sources that can produce singly-charged Cations, including chemical, electron and electrospray. These reactions are similar to the previously described reaction kinetics. The most common reagent cations are benzo(f),quinolone and the noble gases, argon, and xenon. Multiply-charged proteins with opposite polarities have been also reacted along with multiply-charged anions derived from nucleic acid using the multiply charged cations of protein.
“In step 314 (FIG. “In step 314 of the method 300 (FIG. In order to determine the mass spectrum, it is possible to detect ions that have been sequentially ejected in the linear or 3D ion trap according their m/z ratios. Alternately, the ions can be directed to another mass analyzer of a mass spectrometer such as a Time-of-Flight mass analyzer (TOF) or an Orbitrap-type electrostatic trap mass analyser to allow for greater accuracy and resolution than what may be possible by sequential scanning the ion trap. The ion trap can be refilled with new precursor ions by sending the product ions to another analyzer while mass analysis is underway. An Orbitrap or FT-ICR mass analyser are examples of the type of mass analyzer that can be used. If the mass analyzer detects image currents generated by cyclic Ion Motion within an iontrap, then the PTR reactions steps may be beneficially reduced collision cross sections of targeted proteins or polypeptide molecules so that they remain stable in the trap for sufficient time to produce high-quality mass spectrumtra. The PTR productions will also have lower kinetic energy as they leave the high pressure C trap region after their transfer to Orbitrap. mass analyzer. The PTR process will result in the complete desolvation of the product ion population, which will enhance the quality and quantity of the mass spectrum.
“The PTR process generates m/z numbers or alternatively, molecular masses from PTR productions. These can be compared to a database that contains individual pathogen standards. The database will contain patient samples and known reference standards. A small number of pathogen identifications can be obtained by matching the m/z or molecular weights to a database that contains individual referenced pathogens. You can limit the subset by using a specific mass accuracy, weighting individual peak intensities, or weighting molecular weight values according to mass in a scoring system. FIG. 402 illustrates this. 3A. 3A. The m/z molecular information will in all likelihood reduce the number of pathogens that can be unambiguously identified by tandem mass spectrometry. This procedure was described in the international (PCT), patent publication WO 2013/166169. To further refine the identification, Bayesian, logistic regression or decision tree-based methods may be used. This m/z (or molecular weight) search can be performed during data acquisition, i.e. while the sample is being analysed. The search can also be done post-acquisition, i.e. after the sample has been analysed. A comparison of five to ten candidate pathogens with a few molecular weights or m/z values (3-10) of proteins will usually be enough to reduce the number. This is illustrated in FIG. 3A.”
“FIG. 3B illustrates schematically a flow diagram for a second exemplary way, method 370, according to the present teachings. FIG. 3B shows the steps 302-314 in the method 370. The steps 302-314 of the method 370 (FIG. 3A), and so the description of these steps will not be repeated. Only the steps that follow the generation of a mass spectrum at step 314, is where the method 370 differs from method 300. This mass spectrum of PTR productions, as described in the method 300, is sufficient to identify and quantify the proteins and polypeptides of your interest. Tandem mass spectrometry, sometimes referred to by MS/MS and MSn, may be required in some cases to resolve any ambiguities in the recognition or quantification of specific proteins or polypeptide molecules. In these cases, PTR reaction products could be considered to consist of a first-generation reaction product which is then fragmented to create a second generation ions. Combining a specific ratio of first-generation reaction products with one or more specific ratios of fragmentions can often allow for identification of specific proteins or polypeptide molecules associated with pathogens. Many times, the same protein that is identified as a pathogen can also be found in similar pathogens. Method 370, specifically tandem mass spectrometry, may be used to identify a single pathogen. It can be used to analyze as many proteins present in a particular PTR fraction or multiple PTR fractions from the same sample.
“Accordingly, steps 318-322 (FIG. 3B) refers to the application of selected reaction monitoring (SRM), or tandem mass spectrometry, as they are applied to the ions created by PTR. If permitted by the mass spectrometry system, some of the PTR productions may already have been stored (immediately following step 312) in an Ion Storage Apparatus of the mass-spectrometer system. The branching step 315 triggers step 317a. In this case, the previously stored ions can be retrieved for further processing. If the previous batch of PTR productions has been exhausted by the mass analysis (step 314) then the alternative step 317b may be required to be executed again to create a new batch.
“In step 318, certain PTR reaction-productions (i.e. the first-generation productions) within a specific m/z range are mass isolated by ejecting any ions whose ratios are not within that range or ranges. In step 320, the isolated ions are then fragmented. The details of the identified charge-state sequence will be used to determine the range or ranges that are chosen. This will be done automatically by the computer. This is known as “data-dependent analysis”. (or ?data-dependent acquisition?, etc.).”
“Data-dependent fragmentation is used in most conventional MS/MS analyses that involve low-mass molecules, usually a few hundred to several thousand Daltons. It involves choosing the highest P number of most abundant precursors. For tandem mass analysis, the data from a previous MS1 data acquisition is used. The number P can be either a constant input or variable input by the user. This data-dependent analysis is not well-suited for multicomponent biopolymer samples. FIG. FIG. 7C shows two charge state distributions. The envelope 905 and envelope 906 denote the respective envelopes. Each envelope corresponds to a different analyte species in this example. Thus, the sets of lines encompassed by envelopes 905 and 906 may be referred to as ?molecular-species-correlative charge-state distributions?. Consider the FIG. 7C is used to represent precursorions. If P=10 the conventional data-dependent fragmentation method would select the ten most solid vertical lines below the envelope 906 for fragmentation. The conventional technique would not choose any of the dotted lines that correspond to envelope 905 The conventional procedure would therefore yield redundant information about the molecule species that corresponds to envelope 906 but not information regarding the molecule species that corresponds to envelope 905.”
“To overcome the shortcomings of conventional data-dependent fragmentation when applied to high-molecular-weight molecules, the inventors have developed the herein-used novel ?top P unique analyte-specific clusters? data-dependent technique so as to replace, for application to high-molecular-weight molecules, the previous ?top P number of the most abundant precursors? logic. Each molecular-species-correlative charge-state distribution is a set of related mass spectral lines (m/z values) which are interpreted, according to the novel ?top P unique analyte-specific clusters? logic to be all generated from one unique molecule. Each molecular-species-correlative charge-state distribution groups together various charge states and isotopic clusters that are indicated to have been generated from a single molecule, prior to ionization. However, the molecular-species-correlative distribution excludes adducts, which are removed prior to data analysis. According to the novel method, fragmentation is performed only on one (or possibly more) selected representatives of a given molecular-species-correlative charge state distribution envelope thereby avoiding the redundancy noted above associated with the conventional data-dependent fragmentation method. The novel?topP unique analyte specific clusters are described. logic, after a representative m/z ratio (or ratios) has been chosen for a first molecular-species-correlative charge-state distribution, any further fragmentation is directed to a representative m/z ratio of the next determined molecular-species-correlative charge-state distribution, and so on.”
“As described previously, isolation in step 318 of method 370 can be achieved by applying an AC voltage to pairs of electrodes. This will cause ions with m/z values that are outside the range of interest to be ejected from traps while ions that are within the range of ranges or ranges are kept within traps. Sometimes, the ion trap that is used for mass isolation might be the same as the one used to perform the full scan mass analysis in step 314.
The supplemental AC voltage applied in mass isolation to an ion trap may include a summation superimposed frequencies so that ions from two or more non-contiguous ranges of m/z are simultaneously isolated. The mass-isolated first generation product ions are then fragmented using a suitable ion fragmentation method, such as collision-induced dissociation. You can fragment the first-generation productions (products ions formed by PTR or original precursor ions) by transfer them in a known manner to a dedicated fragmentation cells. Here, the transferred ions are broken down so that they generate fragment ions. These fragment ions comprise a second generation reaction products. Optionally, some of the fragment productions can be stored for future fragmentation in optional step 321
“In step 322 (FIG. 3B) The fragments created in step 320 are mass-analyzed by a mass analyzer. The mass analyzer must first transfer the second-generation productions to a specific fragmentation cell before performing step 322. An ion trap mass analyser can be used to analyze second-generation productions in step 322. In this case, the mass analyzer used for step 322 could be the same as the one used to perform the full scan mass analysis in step 314. An accurate mass analyzer that can measure mass-to-charge ratios with a precision of 10 ppm?such an FT-ICR or Orbitrap?type electrostatic trap mass analyser?may also be used for step 322.
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