Improvement of gel-separated protein identification by DMF-assisted digestion and peptide recovery after electroblotting.
In-gel digestion of gel-separated proteins is a major route to assist in proteomics-based biological discovery, which, however, is often embarrassed by its inherent limitations such as the low digestion efficiency and the low recovery of proteolytic peptides. For overcoming these limitations, many efforts have been directed at developing alternative methods to avoid the in-digestion. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel-separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (> or =99.8%). Before tryptic digestion, NH(4)HCO(3) buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane-bound substances, the proteins were virtually in-solution digested in DMF-containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane-enriched sample showed that the method was superior to the reported on-membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.
Lin
Yong
Y
Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, P. R. China.
Li
Yan
Y
Liu
Yi
Y
Han
Wenjun
W
He
Quanze
Q
Li
Jianglin
J
Chen
Ping
P
Wang
Xianchun
X
Liang
Songping
S
D000818:Animals; D004126:Dimethylformamide / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006624:Hippocampus / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
D011506:Proteins; D004126:Dimethylformamide; D014357:Trypsin