Improved 2‐nitrobenzenesulfenyl method: optimization of the protocol and improved enrichment for labeled peptides

Abstract

We have developed the NBS (2-nitrobenzenesulfenyl) method, a quantitative proteome analysis method utilizing stable isotope labeling followed by mass spectrometry. The potential of this method was reported previously, and the procedure has now been further optimized. Here, we describe a procedure utilizing urea or guanidine hydrochloride as a protein denaturant, in conjunction with an improved chromatographic enrichment method for the NBS-labeled peptides using a phenyl resin column. By using this new protocol, both sample loss throughout the protocol and the elution of unwanted unlabeled peptides can be minimized, improving the efficiency of the analysis significantly.

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).…”

Potential biological insights revealed by an integrated assessment of proteomic and transcriptomic data in human colorectal cancer

“…This method is based on stable isotope labeling of tryptophan residues by NBS reagents. As previously described (15,24), this novel method has two major advantages: it reduces the number of peptides by selecting NBS-labeled tryptophan-containing peptides from bulk tryptic digests and a special matrix is used for MALDI-TOF MS measurements that can detect NBS-labeled peptides with high sensitivity. Therefore, this method could improve proteome mining by increasing the dynamic range of detection, and it shows potential for quantitative proteome analysis (25,26).…”

“…NBS-labeled samples were then mixed, reduced, alkylated, and digested by trypsin. NBS-labeled peptides were enriched from tryptic digests and fractionated using Phenyl-Sepharose, as previously described (15). The resulting seven fractions were combined into three fractions and subjected to reversed-phase liquid chroma tography (LC-10ADvp mHPLC system; Shimadzu), as previously described (16).…”