Journal
ANALYTICAL BIOCHEMISTRY
Volume 404, Issue 2, Pages 204-210Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.05.013
Keywords
Gel absorption; In-gel digestion; Membrane proteome; Mass spectrometry; Shotgun
Funding
- Ministry of Science and Technology of China [2004CCA00300]
- National High Technology Research and Development Program (or 863 Program) of China [2006AA02Z141]
- National Basic Research Program (or 973 Program) of China [2010CB529800, 2007CB914203]
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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. (C) 2010 Elsevier Inc. All rights reserved.
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