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Mass spectrometric determination of selenenylsulfide linkages in rat selenoprotein P
Author(s) -
Ma Shuguang,
Hill Kristina E.,
Burk Raymond F.,
Caprioli Richard M.
Publication year - 2005
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/jms.801
Subject(s) - chemistry , selenocysteine , mass spectrometry , dithiothreitol , chromatography , tandem mass spectrometry , electrospray ionization , selenoprotein , cysteine , iodoacetamide , thioredoxin reductase , biochemistry , thioredoxin , glutathione , enzyme , glutathione peroxidase
The reversible formation of a selenenylsulfide linkage in mammalian thioredoxin reductase was identified as having a key role in its activity. Identification of selenenylsulfide and/or diselenide linkages is therefore critical to the determination of the structure and function of selenoproteins. A selenopeptide, 298 SGSAITUQCAENLPSLCSUQGLFAEEK 324 (U = selenocysteine), was isolated from a tryptic digest of rat selenoprotein P. Its two cysteine residues and two selenocysteine (Sec) residues were determined to be present in oxidized form by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The selenopeptide was subjected to partial reduction by dithiothreitol with immediate alkylation by iodoacetamide. This process was monitored by MALDI‐TOFMS to determine the number of alkylations that had taken place. The partially reduced and alkylated peptides were then analyzed by nano‐electrospray ionization tandem mass spectrometry and the results indicated that selenenylsulfide linkages Sec304–Cys314 and Cys306–Sec316 were present. It is concluded that selenoprotein P contains these two selenenylsulfide bonds. Copyright © 2005 John Wiley & Sons, Ltd.