HIGH SENSITIVITY QUANTITATION OF PEPTIDES BY MASS SPECTROMETRY

摘要

The instant invention provides an economical flow-through method for determining amount of target proteins in a sample. An antibody preparation (whether polyclonal or monoclonal, or any equivalent specific binding agent) is used to capture and thus enrich a specific monitor peptide (a specific peptide fragment of a protein to be quantitated in a proteolytic digest of a complex protein sample) and an internal standard peptide (the same chemical structure but including stable isotope labels). Upon elution into a suitable mass spectrometer, the natural (sample derived) and internal standard (isotope labeled) peptides are quantitated, and their measured abundance ratio used to calculate the abundance of the monitor peptide, and its parent protein, in the initial sample.

主权项

1. A method for quantifying the amount of at least one target protein in a complex protein sample comprising the steps of:
(a) digesting the complex protein sample to provide peptides, (b) preparing at least one labeled monitor peptide comprising a subsequence of said target protein(s) to provide an internal standard, (c) adding an aliquot of the product of step (b) containing a known amount of labeled synthetic peptide to the product of step (a), (d) loading the product of step (c) onto at least one support which has attached thereto at least one anti-peptide antibody which binds to at least one monitor peptide, (e) washing the support(s) of step (d) to remove unbound peptides, (f) in eluting the monitor peptide(s) from the anti-peptide antibody bound to the support(s), (g) subjecting at least one of the monitor peptides present in the eluate(s) obtained in step (f) to mass spectrometry to determine the amount of monitor peptide(s) in the digest by comparison with the labeled synthetic standard(s).