In-gel derivatization of proteins for cysteine-specific cleavages and their analysis by mass spectrometry.
As a potential tool for proteomics and protein characterization, in-gel cysteine- and arginine-specific cleavage is demonstrated by means of trypsin or endoproteinase Lys-C for six model proteins (lysozyme, alpha-lactalbumin, beta-lactoglobulin, ribonuclease A, albumin, and transferrin), ranging in size from 14 kDa to 79 kDa. Chemical modifications of cysteine (aminoethylation with bromoethylamine or N-(iodoethyl)-trifluoroacetamide, and subsequent guanidination) and lysine (acetylation) prior to tryptic digestion releases peptides delineated by cysteine or arginine residues. Peptide products are analyzed by MALDI-TOF-MS, ESI-MS, and ESI- and MALDI-MS/MS (with a quadrupole time-of-flight instrument). Complications induced by acrylamide alkylations of cysteines were avoided by substituting lower pH bis-tris polyacrylamide gels for tris-glycine. Sequence coverages from 35 to 86% were obtained and amino acid compositions of generated peptides could be confirmed by comprehensive y- and b-ion series. Detailed information about, in particular, cysteine rich proteins after gel electrophoresis were obtained. The chemistries for modification and cleavage specificities at cysteine residues provide an alternative means to characterize and identify proteins separated by gel electrophoresis.
Thevis
Mario
M
Department of Chemistry and Biochemistry, Mass Spectrometry and Proteomics Technology Center, University of California, Los Angeles, California 90095-1570, USA. mthevis@chem.ucla.edu
Ogorzalek Loo
Rachel R
RR
Loo
Joseph A
JA
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005021:Ethylamines / Q000737:chemistry; D005782:Gels; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism*
D005021:Ethylamines; D005782:Gels; D010446:Peptide Fragments; D011506:Proteins; C004504:2-bromoethylamine; D014357:Trypsin; D008239:Lysine; D003545:Cysteine