Targeted online liquid chromatography electron capture dissociation mass spectrometry for the localization of sites of in vivo phosphorylation in human Sprouty2.
We demonstrate a strategy employing collision-induced dissociation for phosphopeptide discovery, followed by targeted electron capture dissociation (ECD) for site localization. The high mass accuracy and low background noise of the ECD mass spectra allow facile sequencing of coeluting isobaric phosphopeptides, with up to two isobaric phosphopeptides sequenced from a single mass spectrum. In contrast to the previously described neutral loss dependent ECD method, targeted ECD allows analysis of both phosphotyrosine peptides and lower abundance phosphopeptides. The approach was applied to phosphorylation analysis of human Sprouty2, a regulator of receptor tyrosine kinase signaling. Fifteen sites of phosphorylation were identified, 11 of which are novel.
Sweet
Steve M M
SM
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Mardakheh
Faraz K
FK
Ryan
Kevin J P
KJ
Langton
Amy J
AJ
Heath
John K
JK
Cooper
Helen J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002853:Chromatography, Liquid; D004583:Electrons; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D012680:Sensitivity and Specificity
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010748:Phosphopeptides; C485561:SPRY2 protein, human