Analyzing a complex sample by MS/MS using isotopically-labeled standards

• US 9,960,027 B2
• Filed: 05/25/2016
• Issued: 05/01/2018
• Est. Priority Date: 05/25/2016
• Status: Active Grant

• Current Assignee: Thermo Finnigan LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Thermo Finnigan LLC (Thermo Fisher Scientific Incorporated)
• Inventors: McAlister, Graeme C., Dixon, Shannon Eliuk, Huguet, Romain M.
• Primary Examiner(s): Ippolito, Nicole
• Assistant Examiner(s): Chang, Hanway
• Application Number:
  US15/164,113
• Publication Number:
  US 20170345632A1
• Time in Patent Office: 706 Days
• Field of Search: 250281, 250282, 250288
• CPC Class Codes: G01N 2570/00 Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
• CPC Class Codes: G01N 30/7233 interfaced to liquid or supercritical fluid chromatograph interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: H01J 49/0031 Step by step routines describing the use of the apparatus H01J49/0081 takes precedence
• CPC Class Codes: H01J 49/0072 by ion/ion reaction, e.g. electron transfer dissociation, proton transfer dissociation
• CPC Class Codes: H01J 49/4215 Quadrupole mass filters H01J49/4225 takes precedence
• CPC Class Codes: H01J 49/425 with a logarithmic radial electric potential, e.g. orbitraps

Abstract

A method and corresponding apparatus are disclosed for analysis of a peptide-containing sample. The sample is prepared by adding isotopically-labeled peptides corresponding to endogenous peptides of interest, and the prepared sample is analyzed by liquid chromatography-mass spectrometry (LCMS). Detection in a high-resolution, accurate mass (HRAM) MS1 spectrum of a precursor ion matching an isotopically-labeled peptide triggers acquisition of an MS/MS spectrum (preferably acquired in an ion trap or other fast mass analyzer) to determine if a product ion is present matching a characteristic product ion (e.g., the y₁ ion) of the isotopically-labeled peptide. If the characteristic product ion is present, then a HRAM MS/MS spectrum is acquired for detection and quantitation of the corresponding endogenous peptide.

Claims

1. A method for analyzing a peptide-containing sample by mass spectrometry, comprising:

(a) preparing the sample for analysis, including adding a plurality of isotopically labeled peptides to the sample, each one of the isotopically labeled peptides producing, when ionized, a labeled peptide precursor ion of characteristic mass-to-charge (m/z) ratio;

(b) chromotographically separating the sample;

(c) ionizing the separated sample to generate sample ions;

(d) performing an MS1 scan of the sample ions;

(e) identifying a precursor ion in the MS1 scan that has a mass-to-charge ratio (m/z) matching an m/z of a labeled peptide precursor ion;

(f) performing a first MS/MS analysis of the identified precursor ion to acquire a first MS/MS spectrum;

(g) determining whether at least one peak is present in the first MS/MS spectrum matches one or more m/z'"'"'s of a characteristic product ion or product ions generated by dissociation of the labeled peptide precursor ion; and

(h) upon determination that the at least one peak is present in the first MS/MS spectrum, performing a second MS/MS analysis to measure an intensity of at least one product ion produced by dissociation of the endogenous peptide ion corresponding to the labeled precursor ion.

2. The method of claim 1, wherein steps (d) and (h) are performed in a first mass analyzer, and step (f) is performed in a second mass analyzer separate from the first mass analyzer.

3. The method of claim 2, wherein the first mass analyzer is an orbital electrostatic trap mass analyzer, and the second mass analyzer is a quadrupole ion trap mass analyzer.

4. The method of claim 2, wherein the second mass analyzer is a quadrupole mass filter.

5. The method of claim 1, wherein the at least one characteristic product ion is exactly one characteristic product ion.

6. The method of claim 5, wherein the one characteristic product ion is the y₁ product ion of the identified labeled peptide precursor ion.

7. The method of claim 1, wherein step (g) comprises determining whether the at least one peak has an intensity exceeding a threshold value.

8. The method of claim 1, wherein step (g) comprising determining whether the product of the intensity of the at least one peak and the measured intensity of the precursor ion measured in step (d) exceeds a threshold.

9. The method of claim 1, wherein step (g) comprises determining whether a ratio of the intensity of the at least one peak to the measured intensity of the precursor ion measured in step (d) is within a predetermined range of values.

10. The method of claim 1, wherein step (h) further comprises measuring an intensity of at least one product ion produced by dissociation of the identified precursor ion in a third MS/MS analysis.

11. The method of claim 10, wherein the product ion populations produced by the second and third MS/MS analyses are analyzed concurrently.

12. The method of claim 1, wherein step (f) is performed over a narrow m/z window in which the m/z of the at least one characteristic product ion is located.

13. The method of claim 1, wherein step (e) comprises determining whether an ion species present in a spectrum produced in the MS1 scan matches a value on a stored inclusion list.

14. The method of claim 13, wherein the stored inclusion list is adjusted based on chromatographic retention time.

15. The method of claim 1, further comprising:

(i) quantifying the endogenous peptide based on a chromatogram corresponding to the measured intensities of the at least one product ion produced by dissociation of the endogenous peptide.

16. A mass spectrometer configured for mass analysis of a chromatographically separated sample containing a plurality of isotopically labeled peptides, comprising:

an ion source for ionizing the sample to produce sample ions;

a first mass analyzer;

a second mass analyzer, separate and distinct from the first mass analyzer; and

a controller, programmed with instructions for executing the steps of;

(i) causing the first mass analyzer to perform an MS1 scan of the sample ions;

(ii) identifying a labeled peptide precursor ion in the MS1 scan;

(iii) causing the second mass analyzer to perform an MS/MS scan to measure an intensity of at least one characteristic product ion produced by dissociation of the identified labeled peptide precursor ion;

(iv) determining whether the at least one characteristic product ion is present in the MS/MS scan; and

(v) upon determination that the at least one characteristic product ion is present, causing the second mass analyzer to perform a second MS/MS scan to measure an intensity of at least one product ion produced by dissociation of the endogenous peptide ion corresponding to the identified labeled precursor ion.