An approach to quantitative proteome analysis by labeling tryptophan residues

Abstract

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.

Quantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinoma

“…Thus, this method is expected to sensitively detect differences in protein expression with only small samples by increasing the dynamic range of detection. The good reproducibility and reliability of the NBS method have been shown previously [7,8,11,12]. Thus, the method has been demonstrated as promising for nongel-based quantitative profiling.…”

“…Recently, we have developed and improved a quantitative proteomic analysis using 2-nitrobenezenefulfenyl (NBS) reagent, labeled with 12 C 6 (light) or 13 C 6 (heavy) [7][8][9]. Selective introduction of the NBS moiety into tryptophan residues can be achieved and a 6 Da mass difference is generated.…”

Nitrosative protein tyrosine modifications: biochemistry and functional significance

“…The fluorous peptide subset was readily separated from other mixture components by a fluorous-functionalized silica gel because of strong fluorine-fluorine interactions (41). Similarly, enrichment strategies can target the chemical reactivities of additional amino acid side chains (42,43). The combined fractional diagonal chromatography (COFRADIC) approach is based on the concept of diagonal chromatography, in which the collected fraction of the 'primary' run is subjected to chemical or enzymatic modifications of certain residues, thereby changing the polarity of the peptides (44,45).…”

Quantitative protein analysis using 13C7-labeled iodoacetanilide and d5-labeled N-ethylmaleimide by nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry

“…[29][30][31][32][33] Additional methods by labeling other functional groups have also been reported. [34][35][36][37][38] However, as these reagents do not modify more reactive cysteine residues, an additional alkylation process is required.…”