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On‐line derivatization of peptides for improved sequence analysis by micro‐column liquid chromatography coupled with electrospray ionization‐tandem mass spectrometry
Author(s) -
Cárdenas M. S.,
van der Heeft E.,
de Jong A. P. J. M.
Publication year - 1997
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(199708)11:12<1271::aid-rcm925>3.0.co;2-s
Subject(s) - chemistry , chromatography , derivatization , electrospray ionization , tandem mass spectrometry , electrospray , reagent , mass spectrometry , selected reaction monitoring , isobaric labeling , fragmentation (computing) , aqueous solution , protein mass spectrometry , organic chemistry , computer science , operating system
Abstract An on‐line method has been developed for the derivatization and coupled liquid chromatogrpahy (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N ‐succinimidyl‐2(3‐pyridyl)acetate (SPA) in buffered aqueous medium at pH 7 following loading on a precolumn (PC) in a microcolumn switching system. The fast‐hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N ‐pyridylacetyl (PA) derivatives formed are transferred to the analytical micro‐high‐performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N ‐terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N ‐terminal PA group was able to alter the collision‐induced fragmentation pathway of peptides in favour of the formation of abundant b‐type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments. © 1997 John Wiley & Sons, Ltd.