Analysis of integral membrane proteins by heat gel-embedment combined with improved in-gel digestions.
Analysis of integral membrane proteins (IMPs) presents a special challenge because of their hydrophobic nature and low abundance. Here, a new method was developed, which involved heat gel-embedment and improved in-gel digestion of the proteins. Membrane protein lysate containing detergents was mixed with acrylamide solution and the proteins were embedded when the gel polymerized. For comparison, the protein embedment was made at different temperatures (25, 35 or 45 degrees C), and the in-gel digestions were performed in the presence of 0.1% RapiGest reagent (ALS), 0.1% sodium deoxycholate and 10% ACN, respectively. The resultant peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry. Compared with that at 25 degrees C, gel-embedment at 45 degrees C improved the protein embedment and thus protein identification, with the identified IMPs increased by 27%. 0.1% sodium deoxycholate was more efficient than 0.1% ALS and 10% ACN in terms of improving the digestion and tryptic digest recovery of the gel-embedded proteins particularly the hydrophobic IMPs. Out of the 326 IMPs identified by heat gel-embedment combined with improved in-gel digestion strategies, 149 (46%) proteins had at least two mapped transmembrane domains. These results indicate that our newly developed protocol could facilitate the high throughput analysis of integral membrane proteome.
Zhou
Jian
J
The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, PR China.
Huang
Sha
S
Bi
Deng
D
Zhang
Hai
H
Li
Jianglin
J
Lin
Yong
Y
Chen
Ping
P
Wang
Xianchun
X
Liang
Songping
S
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D019295:Computational Biology / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008099:Liver / Q000737:chemistry; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism
D008565:Membrane Proteins; D010446:Peptide Fragments; D011108:Polymers; D014357:Trypsin