Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry |
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Authors: | Jian Zhou Jixian Xiong Jianglin Li Hai Zhang Yong Lin Xianchun Wang Songping Liang |
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Institution: | a 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 b Institute of Neuroscience, Chongqing Medical University, Chongqing 400016, People’s Republic of China |
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Abstract: | 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. |
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Keywords: | Gel absorption In-gel digestion Membrane proteome Mass spectrometry Shotgun |
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