Quantitative proteome analysis of cisplatin-induced apoptotic Jurkat T cells by stable isotope labeling with amino acids in cell culture, SDS-PAGE, and LC-MALDI-TOF/TOF MS.
Quantitative proteome analysis of cisplatin-induced apoptosis in total Jurkat T cell lysates was performed in order to identify modified proteins. Proteins were labeled in cell culture with stable isotopes of arginines, and fractionated by SDS-PAGE. Subsequently, tryptic peptides were analyzed by nano-LC coupled offline to MALDI-TOF/TOF-MS as an alternative to commonly used online LC-ESI-MS. As a result, 26 proteins were found with a relative abundance higher than 1.5, thereof 19 already known and seven unknown to be involved in apoptosis (adenine phosphoribosyltransferase, microsomal signal peptidase 25 kDa subunit, phosphomevalonate kinase, probable rRNA processing protein EBP2, RNA-binding protein 4, transmembrane protein 33, and tetratricopeptide repeat domain 9C). Immunoblotting of core-binding factor beta and elongation factor 2 revealed similar quantitative changes as detected by the SILAC-based proteomics approach. Strikingly, 8 of 26 identified apoptosis-modified proteins contained at least one RNA-binding motif. Three caspase cleavage sites of the 54 kDa nuclear RNA-binding protein (p54nrb) were mapped at DQLD(231) (downward arrow)D, DQVD(286) (downward arrow)R, and MMPD(422) (downward arrow)G by applying caspase-3 to the in vitro translated protein and mutation analysis. The determined caspase cleavage sites were located C-terminal to the two RNA-binding motifs and one (DQLD(231) (downward arrow)D) within the NOPS domain of p54nrb. Concisely, quantitative protein data generated by offline LC-MALDI-MS were shown to be particularly accurate. Furthermore, only regulated peptides were selected in a result-dependent manner for MS/MS analyses and revealed novel apoptosis-modified proteins.
Schmidt
Frank
F
The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
Hustoft
Hanne K
HK
Strozynski
Margarita
M
Dimmler
Christiane
C
Rudel
Thomas
T
Thiede
Bernd
B
D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D017209:Apoptosis / Q000235:genetics*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D018929:Cell Culture Techniques; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002945:Cisplatin / Q000494:pharmacology; D050658:Core Binding Factor beta Subunit / Q000378:metabolism; D004268:DNA-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D019169:Jurkat Cells / Q000378:metabolism; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D034521:Nuclear Matrix-Associated Proteins / Q000378:metabolism*; D050810:Octamer Transcription Factors / Q000378:metabolism*; D020652:Peptide Elongation Factor 2 / Q000378:metabolism; D010449:Peptide Mapping; D020543:Proteome / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000596:Amino Acids; D050658:Core Binding Factor beta Subunit; D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D020652:Peptide Elongation Factor 2; D020543:Proteome; D016601:RNA-Binding Proteins; D001120:Arginine; D008715:Methionine; D053148:Caspase 3; D002945:Cisplatin