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A new approach for sequencing human IRS1 phosphotyrosine‐containing peptides using CapLC‐Q‐TOF micro
Author(s) -
Lu XiaoMing,
Lu Mary Y.,
Fischman Alan J.,
Tompkins Ronald G.
Publication year - 2005
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.826
Subject(s) - chemistry , phosphorylation , peptide , mass spectrometry , chromatography , sh2 domain , tandem mass spectrometry , tyrosine phosphorylation , electrospray , bovine serum albumin , biochemistry , tyrosine
Abstract Reversible phosphorylation of proteins functions as a biological switching network for activation and inhibition of downstream biological processes. Since phosphorylations of these sites are often transient processes, and hence sub‐stoichiometric, systematic characterization of phosphorylation sites is a formidable challenge. In this work, a new approach was developed to pinpoint phosphotyrosine sites on tyrosine‐containing peptides. This required (1) the development of a new and highly sensitive nano‐electrospray assembly and (2) validation of the concept that the specificity and detection limit for trace levels of phosphotyrosine immonium ion in peptide mixtures from protein digests can be increased by varying the collision energy. With our method, an automatic tandem mass spectrometric analysis of peptides eluted from a C 18 capillary liquid chromatographic column is triggered by a positive confirmation of phosphotyrosine immonium ion in a time‐of‐flight mass spectrometric survey. The approach was tested by analyzing the phosphorylation of human IRS‐1 peptides that interact with the Src‐homology 2 domain and mixtures of these peptides with tryptic digests of bovine serum albumin and horse heart myoglobin. Copyright © 2005 John Wiley & Sons, Ltd.