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Variation and quantification among a target set of phosphopeptides in human plasma by multiple reaction monitoring and SWATH‐MS2 data‐independent acquisition
Author(s) -
Zawadzka Anna M.,
Schilling Birgit,
Held Jason M.,
Sahu Alexandria K.,
Cusack Michael P.,
Drake Penelope M.,
Fisher Susan J.,
Gibson Bradford W.
Publication year - 2014
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201400167
Subject(s) - phosphopeptide , phosphorylation , chemistry , phosphoproteomics , proteomics , selected reaction monitoring , human plasma , lipidomics , computational biology , protein phosphorylation , biochemistry , biology , mass spectrometry , tandem mass spectrometry , chromatography , protein kinase a , gene
Human plasma contains proteins that reflect overall health and represents a rich source of proteins for identifying and understanding disease pathophysiology. However, few studies have investigated changes in plasma phosphoproteins. In addition, little is known about the normal variations in these phosphoproteins, especially with respect to specific sites of modification. To address these questions, we evaluated variability in plasma protein phosphorylation in healthy individuals using multiple reaction monitoring (MRM) and SWATH‐MS2 data‐independent acquisition. First, we developed a discovery workflow for phosphopeptide enrichment from plasma and identified targets for MRM assays. Next, we analyzed plasma from healthy donors using an analytical workflow consisting of MRM and SWATH‐MS2 that targeted phosphopeptides from 58 and 68 phosphoproteins, respectively. These two methods produced similar results showing low variability in 13 phosphosites from 10 phosphoproteins (CV inter < 30%) and high interpersonal variation of 16 phosphosites from 14 phosphoproteins (CV inter > 30%). Moreover, these phosphopeptides originate from phosphoproteins involved in cellular processes governing homeostasis, immune response, cell–extracellular matrix interactions, lipid and sugar metabolism, and cell signaling. This limited assessment of technical and biological variability in phosphopeptides generated from plasma phosphoproteins among healthy volunteers constitutes a reference for future studies that target protein phosphorylation as biomarkers.