Isotopic recoding for targeted tandem mass spectrometry

摘要

Aspects of the present disclosure include methods for detecting a low abundance protein and methods for identifying a site of N-glycosylation on a protein. In practicing methods according to certain embodiments, a eukaryotic cell is contacted with an isotopic labeling composition and isotopically labeled N-glycosylated peptides obtained from the eukaryotic cell are assessed by liquid chromatography-tandem mass spectrometry. A predetermined isotopic pattern in the mass spectrum is identified and amino acid sequences of the peptides containing the predetermined isotopic pattern are determined. Systems for identifying a predetermined isotopic pattern in mass spectra and determining amino acid sequences of peptides containing the predetermined isotopic pattern are also described.

主权项

1. A method for identifying a site of N-glycosylation on a protein, the method comprising:
incubating a eukaryotic cell with an isotopic labeling composition comprising two or more different isotopic labels to obtain an isotopically labeled sample of N-glycosylated proteins; enzymatically producing peptides from the isotopically labeled sample of N-glycosylated proteins to obtain an enzymatically treated sample of isotopically labeled peptides; subjecting the enzymatically treated sample of isotopically labeled peptides to liquid-chromatography-tandem mass spectrometry; identifying a predetermined isotopic pattern of the enzymatically treated, isotopically labeled sample of peptides in a mass spectrum at one or more retention times; determining an amino acid sequence of an isotopically labeled peptide present at the one or more retention times; and identifying the site of N-glycosylation on the isotopically labeled protein based on the determined amino acid sequence.