title: Mass spectrometric determination of selenenylsulfide linkages in rat selenoprotein P.
abstract: 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.
authors.affiliation: Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Shuguang
authors.initials: S
authors.lastname: Ma
authors.forename: Kristina E
authors.initials: KE
authors.lastname: Hill
authors.forename: Raymond F
authors.initials: RF
authors.lastname: Burk
authors.forename: Richard M
authors.initials: RM
authors.lastname: Caprioli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D012643:Selenium / Q000032:analysis / Q000737:chemistry*; D051149:Selenoprotein P; D051140:Selenoproteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013440:Sulfides / Q000032:analysis / Q000737:chemistry*
chemical_list: D011506:Proteins; D051149:Selenoprotein P; D051140:Selenoproteins; D013440:Sulfides; D012643:Selenium