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Site‐specific degree of phosphorylation in proteins measured by liquid chromatography‐electrospray mass spectrometry
Author(s) -
Boehm Martin E.,
Seidler Joerg,
Hahn Bettina,
Lehmann Wolf D.
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100561
Subject(s) - phosphopeptide , phosphorylation , protein phosphorylation , chemistry , mass spectrometry , peptide , proteomics , quantitative proteomics , chromatography , isobaric labeling , electrospray , tandem mass spectrometry , computational biology , biochemistry , protein mass spectrometry , biology , protein kinase a , gene
This review focuses on quantitative protein phosphorylation analysis based on coverage of both the phosphorylated and nonphosphorylated forms. In this way, site‐specific data on the degree of phosphorylation can be measured, generating the most detailed level of phosphorylation status analysis of proteins. To highlight the experimental challenges in this type of quantitative protein phosphorylation analysis, we discuss the typical workflows for mass spectrometry‐based proteomics with a focus on the quantitative analysis of peptide/phosphopeptide ratios. We review workflows for measuring site‐specific degrees of phosphorylation including the label‐free approach, differential stable isotope labeling of analytes, and methods based on the addition of stable isotope labeled peptide/phosphopeptide pairs as internal standards. The discussion also includes the determination of phosphopeptide isoform abundance data for multiply phosphorylated motifs that contain information about the connectivity of phosphorylation events. The review closes with a prospective on the use of intact stable isotope labeled proteins as internal standards and a summarizing discussion of the typical accuracies of the individual methods.