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J Proteome Res. 2017 Feb 3;16(2):1069-1076. doi: 10.1021/acs.jproteome.6b00487. Epub 2016 Dec 30.

Neutral Loss Is a Very Common Occurrence in Phosphotyrosine-Containing Peptides Labeled with Isobaric Tags.

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Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Laboratory of Systems Pharmacology, Harvard Medical School , Boston, Massachusetts 02115 United States.
Cell Signaling Technology, Inc. , Danvers, Massachusetts 01923, United States.


While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.


high-energy collision-induced dissociation; immonium ion; isobaric tag; neutral loss; phosphorylation; phosphotyrosine; proton mobility; site localization; tandem mass tag

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