On-target sample preparation of 4-sulfophenyl isothiocyanate-derivatized peptides using AnchorChip Targets.
De novo sequencing of tryptic peptides by post source decay (PSD) or collision induced dissociation (CID) analysis using MALDI TOF-TOF instruments is due to the easy interpretation facilitated by the introduction of N-terminal sulfonated derivatives. Recently, a stable and cheap reagent, 4-sulfophenyl isothiocyanate (SPITC), has been successfully used for N-terminal derivatization. Previously described methods have always used desalting and concentration by reverse-phase chromatography prior to mass spectrometric analysis. Here we present an on-target sample preparation method based on AnchorChip target technology. The method was optimized for reduction of by-products and sensitivity with SPITC-derivatized tryptic BSA peptides, and successfully applied to protein identification from silver-stained two-dimensional electrophoretic gels of fish liver extracts. The method is simple and sensitive and allowed protein identification based on de novo sequencing and BLAST search from species with limited sequence information.
Zhang
Xumin
X
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK 5230 Odense M, Denmark.
Rogowska-Wrzesinska
Adelina
A
Roepstorff
Peter
P
D000097:Acetonitriles / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry*; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D029941:Fish Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D005432:Flounder; D017879:Isothiocyanates / Q000737:chemistry*; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D000097:Acetonitriles; D001557:Benzenesulfonates; D029941:Fish Proteins; D017879:Isothiocyanates; D010455:Peptides; C015308:4-isothiocyanatobenzenesulfonate; D014269:Trifluoroacetic Acid; D014357:Trypsin; C032159:acetonitrile