Shotgun analysis of membrane proteomes using a novel combinative strategy of solution-based sample preparation coupled with liquid chromatography-tandem mass spectrometry.
Although much effort has been made in the field of membrane proteomics, the analysis of membrane proteins particularly integral membrane proteins with poor water solubility still presents a great challenge. In this paper, 2% SDS was used to extract membrane proteins and experimental conditions for the application of acetone precipitation method to the cleanup of SDS-solubilized membrane protein sample were optimized. For improving the re-dissolution and trypsinolysis of acetone-precipitated proteins, several commonly used additives, urea, methanol and sodium deoxycholate (SDC), were employed and compared. The results showed that, when the pre-cooled acetone-to-sample ratio was 6:1 (v/v) with one additional washing step, residual SDS in the protein sample could be lowered to below 0.01% and more than 90% of the proteins were precipitated and therefore recovered. 1% SDC-containing buffer could improve the re-dissolution and digestion of the acetone precipitated proteins more efficiently than the others. Using the combinative sample preparation strategy developed, 398 proteins were identified from the rat liver membrane-enriched fraction, including 188 membrane proteins. Compared with other three representative solution-based sample preparation methods commonly used in membrane proteomics, the newly developed combinative strategy increased the number of identified total proteins and membrane proteins on average by 29.2% and 28.5%, respectively. This combinative strategy was demonstrated to be easily operated at low cost and suitable for the analysis of membrane proteins varying in type and sample volume, etc.
Key Laboratory of Protein Chemistry and Developmental Biology of National Education Committee, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, PR China.
Yong
Y
Lin
Hui
H
Liu
Zhonghua
Z
Liu
Xianchun
X
Wang
Songping
S
Liang
D000096:Acetone / Q000737:chemistry; D000818:Animals; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000379:methods*; D008099:Liver / Q000737:chemistry / Q000166:cytology; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012996:Solutions; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry
D008565:Membrane Proteins; D020543:Proteome; D012996:Solutions; D000096:Acetone; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin