Improved 2-nitrobenzenesulfenyl method: optimization of the protocol and improved enrichment for labeled peptides.
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.
Matsuo
Ei-Ichi
E
Life Science Laboratory, Shimadzu Corporation, Kyoto 604-8511, Japan.
Toda
Chikako
C
Watanabe
Makoto
M
Iida
Tetsuo
T
Masuda
Taro
T
Minohata
Toshikazu
T
Ando
Eiji
E
Tsunasawa
Susumu
S
Nishimura
Osamu
O
D000818:Animals; D002645:Chickens; D019791:Guanidine; D051379:Mice; D009578:Nitrobenzenes / Q000737:chemistry*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D011489:Protein Denaturation; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein; D014508:Urea
D009578:Nitrobenzenes; D010455:Peptides; C002732:2-nitrobenzenesulfenyl chloride; D014508:Urea; D019791:Guanidine