Development of a highly efficient 2-D system with a serially coupled long column and its application in identification of rat brain integral membrane proteins with ionic liquids-assisted solubilization and digestion.
Two dimensional high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (2D-HPLC-ESI-MS/MS) is one of the most powerful techniques for high resolution, efficiency, and throughput separation and identification of proteomes. For a bottom-up strategy-based proteome analysis, usually multistep salt elution was needed in the first dimension separation by SCX, to simplify the peptides for the further second dimensional separation by RPLC. Here, by using a 30 cm-long serially coupled long column (SCLC) in the second dimension, we reduced the salt steps of SCX from 13 to 5 to shorten the total analysis time. Compared to the commonly applied 2D-HPLC with over 10-step salt elution in SCX and microRPLC with a short column (SC), named as SC-2D, the peak capacity of 2D-HPLC with a SCLC column, named as SCLC-2D, was increased 3.3-folds while the analysis time was increased by only 1.17-folds. Therefore, the time-based protein identification efficiency was ∼55 protein groups/h, nearly 2-fold of that for SC-2D (∼28 protein groups/h). With the further combination of assisted solubilization by ionic liquids and SCLC-2D, 608 integral membrane proteins (IMPs) (27.66% of the total 2198 proteins, FDR < 1%) were identified from rat brain, more than those obtained by the traditional urea method (252 unique IMPs, occupying 17.03% of total 1480 proteins). All of these results demonstrate the promise of the developed technique for large-scale proteome analysis.
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, People's Republic of China.
Dingyin
D
Tao
Xiaoqiang
X
Qiao
Liangliang
L
Sun
Chunyan
C
Hou
Liang
L
Gao
Lihua
L
Zhang
Yichu
Y
Shan
Zhen
Z
Liang
Yukui
Y
Zhang
D000085:Acetates / Q000737:chemistry; D000097:Acetonitriles / Q000737:chemistry; D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002412:Cations / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D005561:Formates / Q000737:chemistry; D052578:Ionic Liquids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
D000085:Acetates; D000097:Acetonitriles; D002412:Cations; D005561:Formates; D052578:Ionic Liquids; D008565:Membrane Proteins; C030544:formic acid; C018824:ammonium acetate; C032159:acetonitrile