Search

US-20260126413-A1 - QUANTITATIVE SHOTGUN PROTEOME, LIPIDOME, AND METABOLOME ANALYSIS BY DIRECT INFUSION

US20260126413A1US 20260126413 A1US20260126413 A1US 20260126413A1US-20260126413-A1

Abstract

The present invention provides methods and systems using gas-phase separation with mass spectrometry analysis instead of liquid chromatography, thereby enabling faster peptide, proteome, and multi-omic analysis. Also provided are improved methods and software for data independent acquisition. One embodiment referred to as Direct Infusion—Shotgun Proteome Analysis (DI-SPA) used with data-independent acquisition mass spectrometry (DIA-MS), resulted in targeted quantification of over 500 proteins within minutes of MS data collection (˜3.5 proteins/second). Enabling fast, unbiased protein and proteome quantification without liquid chromatography, DI-SPA offers a new approach to boosting throughput critical to drug and biomarker discovery studies that require analysis of thousands of proteomes. This invention is also able to perform complex multi-omic analysis of proteomes, lipidomes, and metabolomes on a single platform.

Inventors

  • Jesse Meyer
  • Joshua Coon
  • Alexander HEBERT
  • Caleb CRANNEY

Assignees

  • WISCONSIN ALUMNI RESEARCH FOUNDATION
  • THE MEDICAL COLLEGE OF WISCONSIN, INC.

Dates

Publication Date
20260507
Application Date
20260105

Claims (20)

  1. 1 . A method for high-throughput analysis of a sample using mass spectrometry, the method comprising the steps of: a) providing a sample comprising a mixture of molecules; b) ionizing the mixture of molecules thereby generating ionized molecules; c) transporting the ionized molecules from an inlet side of a chamber to an outlet side of a chamber, wherein the chamber comprises a buffer gas and the ionized molecules are transported through the buffer gas; d) applying an electric field to the ionized molecules being transported through the buffer gas, and separating the ionized molecules according to ion mobility of the ionized molecules; e) transporting a portion of the separated ionized molecules through the outlet side of the chamber into a mass analyzer of a mass spectrometer device, wherein the chamber is in fluid connection with the mass analyzer; f) selectively isolating the portion of the separated ionized molecules in the mass analyzer according to the mass-to-charge ratios of the separated ionized molecules, thereby generating isolated ionized molecules; and g) measuring mass-to-charge ratios of the isolated ionized molecules, thereby generating mass spectrometry data.
  2. 2 . The method of claim 1 , wherein the mixture comprises 1,000 or more target species of molecules and the method is able to generate mass spectrometry data from each of the 1,000 or more target species within one hour.
  3. 3 . The method of claim 1 , wherein liquid chromatography is not performed on the mixture of molecules.
  4. 4 . The method of claim 1 , wherein an online separation or purification step is not performed on the mixture of molecules or ionized molecules other than the ion mobility separation.
  5. 5 . The method of claim 1 , wherein the mixture comprises polypeptides, lipids, metabolites, or combinations thereof.
  6. 6 . The method of claim 1 further comprising digesting a protein mixture to generate an unseparated mixture of molecules; and ionizing the unseparated mixture of molecules thereby generating the ionized molecules.
  7. 7 . The method of claim 1 , wherein the mixture of molecules is a whole cell lysate and the step of generating ionized molecules comprises ionizing the whole cell lysate.
  8. 8 . The method of claim 1 further comprising fragmenting the isolated ionized molecules, thereby generating fragment ions, and measuring the mass-to-charge ratios of the fragment ions, wherein the generated mass spectrometry data comprises the mass-to-charge ratios of the fragment ions.
  9. 9 . The method of claim 1 wherein applying the electric field comprises varying the strength of the electric field while the ionized molecules are transported through the chamber.
  10. 10 . The method of claim 1 wherein the generated mass spectrometry data further comprises ion mobility data, wherein the ion mobility data comprises: time when the separated ionized molecules used to generate the mass-to-charge ratios are collected and transported to the mass analyzer, an order separated ions were collected, voltage used to separate the ionized molecules, or combinations thereof.
  11. 11 . The method of claim 10 wherein the generated mass spectrometry data further comprises a generated spectrum, wherein the generated spectrum comprises a plurality of peaks corresponding to measured mass-to-charge ratios of the isolated ionized molecules, and wherein the plurality of peaks are characterized by one or more signal parameters.
  12. 12 . The method of claim 10 further comprising comparing generated mass spectrometry data or spectra with one or more reference mass spectrometry data or spectra from a reference database, and identifying one or more molecules from the sample as corresponding to a compound from the reference database.
  13. 13 . The method of claim 12 comprising comparing the generated spectra with one or more spectra from a reference database, said comparing step comprising: a) assigning a spectrum tag to each peak from at least two selected reference spectra from the one or more reference spectra; b) combining the at least two selected reference spectra to form a consolidated reference spectrum; c) comparing the one or more peaks from the generated spectrum with each peak in the consolidated reference spectrum; and d) identifying a target peak from the consolidated reference spectrum that matches a peak from the generated spectrum using the spectrum tag.
  14. 14 . A method of analyzing a proteome using mass spectrometry, the method comprising the steps of: a) collecting a portion of a proteome from a cell; b) digesting the portion of the proteome to form a mixture of polypeptides; c) ionizing the mixture of polypeptides thereby generating ionized polypeptides; d) transporting the ionized polypeptides from an inlet side of a chamber to an outlet side of a chamber, wherein the chamber comprises a buffer gas and the ionized polypeptides are transported through the buffer gas; e) applying an electric field to the ionized polypeptides being transported through the buffer gas, and separating the ionized polypeptides according to ion mobility of the ionized polypeptides; f) transporting a portion of the separated ionized polypeptides through the outlet side of the chamber into a mass analyzer of a mass spectrometer device, wherein the chamber is in fluid connection with the mass analyzer; g) selectively isolating the portion of the separated ionized polypeptides in the mass analyzer according to the mass-to-charge ratios of the separated ionized polypeptides, thereby generating isolated ionized polypeptides; and h) measuring mass-to-charge ratios the isolated ionized polypeptides, thereby generating mass spectrometry data, wherein an online separation or purification step is not performed on the mixture of molecules or ionized molecules other than the ion mobility separation.
  15. 15 . A method of analyzing a sample comprising one or more molecules using mass spectrometry, the method comprising the steps of: a) introducing the sample to an ionization source, thereby generating one or more ionized molecules; b) performing an ion filtering step on the ionized molecules comprising selectively transmitting a first portion of ionized molecules to a mass analyzer, wherein ionized molecules within the transmitted first portion of ionized molecules have an ion mobility within a first predefined ion mobility range; c) performing a mass filtering step comprising selectively isolating a first distribution of transmitted ions from the transmitted first portion of ionized molecules, wherein the isolated first distribution of transmitted ions have a mass-to-charge ratio within a first predefined mass-to-charge ratio range; d) generating mass spectrometry data comprising recording mass-to-charge ratios of the isolated first distribution of transmitted ions; and e) comparing the generated mass spectrometry data with one or more reference mass spectrometry data from a reference database, and identifying one or more target species of molecules from the sample as corresponding to a compound from the reference database.
  16. 16 . The method of claim 15 further comprising fragmenting the isolated first distribution of transmitted ions, thereby generating first product ions, and recording mass-to-charge ratios of the first product ions, wherein the generated mass spectrometry data comprises the mass-to-charge ratios of the first product ions.
  17. 17 . The method of claim 15 , wherein the ion filtering step further comprises selectively transmitting a second portion of ionized molecules to a mass analyzer, wherein ionized molecules within the second portion of ionized molecules have an ion mobility within a second predefined ion mobility range; wherein the mass filtering step comprises selectively isolating a second distribution of transmitted ions from the transmitted second portion of ionized molecules, wherein the isolated second distribution of transmitted ions have a mass-to-charge ratio within a second predefined mass-to-charge ratio range; and generating mass spectrometry data comprises recording mass-to-charge ratios of the isolated second distribution of transmitted ions.
  18. 18 . The method of claim 15 further comprising enzymatically digesting the sample prior to introducing the sample to the ionization source, wherein liquid chromatography (LC) is not performed on the digested sample.
  19. 19 . The method of claim 15 , wherein an online separation or purification step is not performed on the sample other than the ion filtering step.
  20. 20 . The method of claim 15 wherein the generated mass spectrometry data further comprises ion mobility data, wherein the ion mobility data comprises: time when the separated ionized molecules used to generate the mass-to-charge ratios are collected and transported to the mass analyzer, an order separated ions were collected, voltage used to separate the ionized molecules, or combinations thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 17/741,277, filed May 10, 2022, which claims priority from U.S. Provisional Patent Application No. 63/187,190, filed May 11, 2021, which are incorporated by reference herein to the extent that there is no inconsistency with the present disclosure. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under LM007359 and GM108538 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND OF THE INVENTION Shotgun proteomics methods using liquid chromatography coupled to mass spectrometry (LC-MS) achieve the greatest depth and breadth of proteome coverage (Aebersold et al., Nature, 537, 347-355 (2016); and Meyer et al., Expert Review of Proteomics, 14, 419-429 (2017)). The time required for such comprehensive proteome analysis, once a major burden, has been driven down by technological adaptation. Just over a decade ago, weeks of MS data collection were required to quantify nearly all expressed yeast proteins (de Godoy et al., Nature, 455, 1251-1254 (2008)); by 2016, the same task could be accomplished in just over one hour (see, for example, Hebert et al., Molecular & Cellular Proteomics, 13, 339-347 (2014)). More recent advancements in data-independent acquisition (DIA) and fast LC have further reduced analysis times and enabled routine protein quantification at rates of up to 15,000 non-unique proteins per hour (Bache et al., Molecular & Cellular Proteomics, 17, 2284-2296 (2018) and Kelstrup et al., J. Proteome Res., 17, 727-738 (2018)). Similar methods are also useful for rapid analysis and quantification in lipidomics and metabolomics. Still, as the fields of proteomics, lipidomics, and metabolomics push for higher throughput, the requirement for liquid-phase separations inevitably requires time that in turn limits throughput. This is amplified by time needed to load and re-equilibrate the LC column. In theory, omitting LC prefractionation could decrease analysis time (Gachumi et al., Anal. Chem., 92, 8628-8637 (2020)). Several papers describe qualitative analysis of peptides from simple mixtures by direct infusion, an approach that has been used in metabolomics (Chekmeneva et al., J. Proteome Res., 16, 1646-1658 (2017); and Koulman et al., Rapid Communications in Mass Spectrometry, 21, 421-428 (2007)). Twenty-five years ago, direct infusion of peptides from trypsin-digested gel bands or standard proteins was available, but offered limited depth, typically less than 60 peptides (Wilm et al., Nature, 379, 466-469 (1996); Chen, S., Proteomics, 6, 16-25 (2006); Pereira-Medrano et al., Journal of the American Society for Mass Spectrometry, 18, 1714-1725 (2007); Chen et al., Proteome Science, 9, 38 (2011); Xiang et al., Anal. Chem., 84, 1981-1986 (2012); and Kretschy et al., International Journal of Mass Spectrometry, 307, 105-111 (2011)). As LC and MS co-evolved, LC-MS became the premiere technology for the analysis of the tremendously complex mixture of peptides that results from whole proteome digestion. Although direct infusion was recently used to profile histone modifications in one minute (Sidoli et al., Genome Res., 29, 978-987 (2019)), it has not been able to interrogate peptide mixtures from the human proteome, which contain well over 100,000 distinct peptide sequences (Michalski et al., Journal of Proteome Research, 10, 1785-1793 (2011)). Several factors may hinder detection of peptides from such complex mixtures by electrospray ionization without LC, including: peptide polarity, mobile phase composition, ion suppression, and ion competition (Cech et al., Anal. Chem., 72, 2717-2723 (2000); Meyer et al., Journal of the American Society for Mass Spectrometry, 1-10 (2012); Ogorzalek et al., J. Am. Soc. Mass Spectrom., 25, 1675-1693 (2014); Annesley et al., Clin. Chem., 49, 1041 (2003); and Sarvin et al., Nat Commun., 11, 3186 (2020)). However, recent advancements in MS around accurate mass measurement, sensitivity, and speed inspired the present inventors to revisit the concept of peptide identification without LC. Among recent MS advances, ion mobility has enabled an additional dimension of gas-phase peptide cation separation that complements fractionation by quadrupole selection (see, for example, Webb et al., Anal. Chem., 86, 9169-9176 (2014); Giles et al., Anal. Chem., 91, 8564-8573 (2019); Meier et al., Journal of Proteome Research, 14, 5378-5387 (2015); Swearingen et al., Molecular & Cellular Proteomics, 11, M111.014985 (2012); Hebert et al., Anal. Chem., 90, 9529-9537 (2018); Nagy et al., Anal. Chem., 91, 4374-4380 (2019); Melani et al., bioRxiv, (2019) doi: 10.1101/693473; Purves et al., Journal of The American Society for Mass Spectrometry, 28, 525-538 (2017); Hengel et al., J. Proteome Res., 10, 4567-4578 (2011); McLean et al., Int. J. Mass Spec., 240(3): 301-315; Yi et al., Ele