NDLI: Quantification of Competing H$_{3}$PO$_{4}$ Versus HPO$_{3 }$+ H$_{2}$O Neutral Losses from Regioselective $^{18}$O-Labeled Phosphopeptides

Content Provider	Springer Nature Link
Author	Cui, Li Yapici, Ipek Borhan, Babak Reid, Gavin E.
Copyright Year	2013
Abstract	Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H$_{3}$PO$_{4}$ from the phosphorylated residue and the combined losses of HPO$_{3}$ and H$_{2}$O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS$^{3}$ for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective $^{18}$O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the –100 Da (–H$_{3}$PO$_{3}$ $^{18}$O) versus –98 Da (–[HPO$_{3}$ + H$_{2}$O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups. Figure ᅟ
Starting Page	141
Ending Page	148
Page Count	8
File Format	PDF
ISSN	10440305
Journal	Journal of The American Society for Mass Spectrometry
Volume Number	25
Issue Number	1
e-ISSN	18791123
Language	English
Publisher	Springer US
Publisher Date	2013-11-19
Publisher Institution	The American Society for Mass Spectrometry
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Peptide fragmentation Phosphopeptide MS/MS Isotope labeling Analytical Chemistry Biotechnology Organic Chemistry Proteomics Bioinformatics
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Structural Biology

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