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Simultaneous detection of N‐terminal fragment ions in a protein mixture using a ruthenium(II) complex
Author(s) -
Ito Akihiro,
Okamura Takaaki,
Yamamoto Hitoshi,
Ueyama Norikazu,
Yamaguchi Minoru,
Kuyama Hiroki,
Ando Eiji,
Tsunasawa Susumu,
Ake Kojiro,
Masui Ryoji,
Kuramitsu Seiki,
Nakazawa Takashi,
Norioka Shigemi
Publication year - 2007
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/rcm.3126
Subject(s) - chemistry , ruthenium , thermus thermophilus , mass spectrometry , moiety , chromatography , stereochemistry , biochemistry , escherichia coli , gene , catalysis
Use of a bis(terpyridine)ruthenium(II) derivative as an N‐terminal labeling reagent resulted in the simultaneous detection and individual determination of all the N‐terminal fragments of the proteins in a mixture without requiring any separation. All of the N‐termini of the guanidinated proteins were labeled selectively by the ruthenium complex (‐CO‐labeling). After chymotryptic digestion, the fragments were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) and post‐source decay (PSD). The ‐CO moiety exclusively enhanced N‐terminal fragment ions in mass spectra and enabled easy N‐terminal sequencing. In a mixture containing three different proteins (lysozyme, ubiquitin, and insulin), all of the N‐terminal fragment ions labeled with the ruthenium complex were found to produce uniformly intense peaks without the detection of the other unlabeled fragments. The N‐terminal sequences of these ions were determined individually by PSD analysis. Application to unknown proteins from Thermus thermophilus HB8 with two‐dimensional electrophoretic separation resulted in the successful determination of the N‐terminal sequence and easy identification of the target protein. Copyright © 2007 John Wiley & Sons, Ltd.