Dried polyacrylamide gel absorption: a method for efficient elimination of the interferences from SDS-solubilized protein samples in mass spectrometry-based proteome analysis.
Sample preparation holds an important place in MS-based proteome analysis. For effective proteolysis and MS analysis, it is essential to eliminate the interferences while extracting the analytes of interest from complex mixtures. To address this, herein we describe a new dried polyacrylamide gel absorption method. In this method, the protein sample prepared using high concentration of SDS was directly and completely absorbed by vacuum-dried polyacrylamide gel, and then the interfering substances including SDS and some other salts were efficiently removed by in-gel washing steps while retaining the denatured proteins in the gel, thus offering a clean environment amenable to downstream buffer exchange, proteolytic digestion and digest recovery, etc. In combination with in-gel digestion and LC-MS/MS, the newly developed method was applied to the proteome analyses of membrane-enriched fraction and whole tissue homogenate. It was demonstrated that the method is suitable for the analysis of a complex biological sample and can be widely used for sample cleanup in shotgun proteome analyses.
The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, PR China.
Jian
J
Zhou
Jianglin
J
Li
Jianjun
J
Li
Ping
P
Chen
Xianchun
X
Wang
Songping
S
Liang
D000042:Absorption; D000818:Animals; D002462:Cell Membrane; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006624:Hippocampus / Q000737:chemistry; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012995:Solubility
D010446:Peptide Fragments; D011506:Proteins; D012967:Sodium Dodecyl Sulfate