Absolute quantitation of proteins and protein modifications by mass spectrometry with multiplexed internal standards

摘要

A method for absolute protein or peptide quantitation by mass spectroscopy. A sample containing a protein or peptide of interest is prepared for mass spectroscopy analysis. The sample is subjected to mass spectroscopy analysis at low resolution whereby a single additive mass spectroscopy peak is obtained, then is subjected to high resolution mass spectroscopy analysis whereby a plurality of mass spectroscopy peaks are obtained. The intensity of each of the plurality of mass spectroscopy peaks is quantitated either by comparison to an internal standard set, or by using a standard curve generated for each isotopologue set. Quantitation using a standard curve enhances quantitation across a dynamic range of analyte.

主权项

1. A method for quantitation of a target protein or peptide in a sample by mass spectroscopy (MS), the method comprising
preparing internal standard isotopologues using at least one set of multiplexed heavy peptide internal standards, wherein each peptide within the at least one set contains the same amino acid sequence, but the peptides within the set differ by a mass defect created by incorporating heavy isotopes on different atoms within at least one amino acid molecule resulting in internal standard isotopologues, and quantitating at least one target protein or peptide using the multiplexed internal standard isotopologues.