Analyzing a Complex Sample by MS/MS Using Isotopically-Labeled Standards
First Claim
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) chromatographically 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 matching one or more m/z'"'"'s of a characteristic product ion or product ions generated by dissociation of the labeled peptide precursor ion, wherein the characteristic product ion or product ions consists of no more than three product ions; 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.
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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.
3 Citations
18 Claims
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1. A method for analyzing a peptide-containing sample by mass spectrometry, comprising:
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(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) chromatographically 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 matching one or more m/z'"'"'s of a characteristic product ion or product ions generated by dissociation of the labeled peptide precursor ion, wherein the characteristic product ion or product ions consists of no more than three product ions; 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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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Specification