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Studies on the dephosphorylation of phosphotyrosine‐containing peptides during post‐source decay in matrix‐assisted laser desorption/ionization
Author(s) -
Metzger Sabine,
Hoffmann Ralf
Publication year - 2000
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/1096-9888(200010)35:10<1165::aid-jms44>3.0.co;2-r
Subject(s) - chemistry , dephosphorylation , phosphoserine , phosphorylation , aspartic acid , phosphopeptide , tyrosine , mass spectrometry , amino acid , peptide , biochemistry , phosphoric acid , peptide sequence , chromatography , serine , phosphatase , organic chemistry , gene
Abstract Phosphorylation of tyrosine residues in proteins is a common regulatory mechanism, although it accounts for less than 1% of the total O ‐phosphate content in proteins. Whereas aromatic phosphorylation sites can be identified by a number of different analytical techniques, sequence analysis of phosphotyrosine‐containing proteins at the low picomole or even femtomole level is still a challenging task. This paper describes the post‐source decay in matrix‐assisted laser desorption/ionization mass spectrometry of phosphotyrosine‐containing model peptides by comparing their fragmentation behavior with sequence‐homologous unphosphorylated peptides. Whereas the parent ions showed significant losses of HPO 3 , all phosphorylated fragment ions of the b‐ and y‐series displayed only minor dephosphorylated signals, which often were not detectable. Surprisingly, one of the studied phosphotyrosine‐containing sequences displayed, in addition to the [M + H − 80] + ion, a more abundant [M + H − 98] + ion, which could be explained by elimination of phosphoric acid. This dephosphorylation pattern was very similar to the patterns obtained for phosphoserine‐ and phosphothreonine‐containing peptides. Because the dephosphorylation pattern of the parent ion is often used to identify modified amino acids in peptides, we investigated possible dephosphorylation mechanisms in detail. Therefore, we substituted single trifunctional amino acid residues and incorporated deuterated phosphotyrosine residues. After excluding direct elimination of phosphoric acid from tyrosine, we could show that the obtained loss of H 3 PO 4 depends on aspartic acid and arginine residues. Most likely the HPO 3 group is transferred to aspartic acid followed by cleavage of phosphoric acid forming a succinimide. On the other hand, arginine appears to induce the H 3 PO 4 loss by protonation of phosphotyrosine leaving a phenyl cation. Copyright © 2000 John Wiley & Sons, Ltd. Abbreviations Bzl benzylCD circular dichroismCID collision‐induced dissociationCIF charge‐induced fragmentationCRF charge‐remote fragmentationDE delayed‐extractionDHB 2,5‐dihydroxybenzoic acidESI electrospray ionizationFAB fast‐atom bombardmentFmoc 9‐fluorenylmethoxycarbonylMALDI matrix‐assisted laser desorption/ionizationMS mass spectrometryMS/MS tandem mass spectrometryNMR nuclear magnetic resonancePSD post‐source decayRP‐HPLC reversed‐phase high‐performance liquid chromatographyTyr(P) O ‐phospho‐ L ‐tyrosine.