Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry.
A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.
Zhou
Jian
J
Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, People's Republic of China.
Xiong
Jixian
J
Li
Jianglin
J
Huang
Sha
S
Zhang
Hai
H
He
Quanze
Q
Lin
Yong
Y
Chen
Ping
P
Wang
Xianchun
X
Liang
Songping
S
D000042:Absorption; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D008565:Membrane Proteins; D020543:Proteome; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin