An approach to quantitative proteome analysis by labeling tryptophan residues.
This report describes a method for quantification and sequence identification of individual proteins in complex mixtures. The method is based on labeling with the chemical reagent 2-nitrobenzenesulfenyl chloride (NBSCl) in conjunction with tandem mass spectrometry. In this method, selective introduction of the 2-nitrobenzenesulfenyl (NBS) moiety onto tryptophan residues is achieved, and a 6 Da mass differential is generated using (13)C(6)-labeled NBSCl (NBSCl-(13)C(6)) and (12)C(6)-labeled NBSCl (NBSCl-(12)C(6)). The 6 Da mass differential between the NBS-(12)C(6)-labeled and the NBS-(13)C(6)-labeled peptides assigns a mass signature to all tryptophan-containing peptides in any pool of proteolytic digests for protein identification through peptide mass mapping. Using this strategy, we compared the protein expression in rat sera using a normal (control) rat (Crj:Wistar) and a hyperglycemic rat (GK/Crj). The stable isotope dilution techniques used in this method provide highly accurate relative quantification. The NBS approach offers a widely applicable means of analyzing protein mixtures derived from biological samples, and the method described here presents an effective and simplified approach to proteome analysis.
Kuyama
Hiroki
H
Life Science Laboratory, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan.
Watanabe
Makoto
M
Toda
Chikako
C
Ando
Eiji
E
Tanaka
Koichi
K
Nishimura
Osamu
O
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003366:Cosyntropin / Q000737:chemistry; D019004:Galanin / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007202:Indicators and Reagents; D009578:Nitrobenzenes; D010455:Peptides / Q000737:chemistry; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014364:Tryptophan / Q000032:analysis*
D007202:Indicators and Reagents; D009578:Nitrobenzenes; D010455:Peptides; D020543:Proteome; D003366:Cosyntropin; C002732:2-nitrobenzenesulfenyl chloride; D019004:Galanin; D014364:Tryptophan; D014357:Trypsin