Identification of selenium-containing proteins in selenium-rich yeast aqueous extract by 2D gel electrophoresis, nanoHPLC-ICP MS and nanoHPLC-ESI MS/MS.
An approach based on the consecutive use of nanoHPLC-ICP collision cell MS and nanoHPLC-electrospray MS was proposed for the analysis of water-soluble selenium-containing proteins in selenium-rich yeast after their separation by 2D gel electrophoresis (GE). An ultrasonic probe was employed for fast protein extraction avoiding sample heating and thus reducing the risk of protein degradation. The efficiency of different extraction steps were critically evaluated by total selenium analysis and size-exclusion chromatography (SEC)-ICP MS. Prior to electrophoresis proteins were purified by acetone precipitation. The protein-containing spots from 2D GE were excised and digested with trypsin. The digests obtained were analyzed by nanoHPLC-ICP MS in order to check for the presence of selenium-containing peptides; this allowed the detection of target proteins for further analyses (two out of five spots). The subsequent analyses of the selected digests by nanoHPLC-ES MS/MS allowed the attribution of amino acid sequences to peaks detected by ICP MS revealing the presence of two selenium-containing proteins: SIP 18 and HSP 12.
Tastet
Laure
L
Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, CNRS UMR 5254, Hélioparc, 2 av. Pr. Angot, F-64053 Pau, France.
Schaumlöffel
Dirk
D
Bouyssiere
Brice
B
Lobinski
Ryszard
R
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012643:Selenium / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D014867:Water / Q000737:chemistry*
D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; D014357:Trypsin; D012643:Selenium