title: Biphasic microreactor for efficient membrane protein pretreatment with a combination of formic acid assisted solubilization, on-column pH adjustment, reduction, alkylation, and tryptic digestion.
abstract: Combining good dissolving ability of formic acid (FA) for membrane proteins and excellent complementary retention behavior of proteins on strong cation exchange (SCX) and strong anion exchange (SAX) materials, a biphasic microreactor was established to pretreat membrane proteins at microgram and even nanogram levels. With membrane proteins solubilized by FA, all of the proteomics sample processing procedures, including protein preconcentration, pH adjustment, reduction, and alkylation, as well as tryptic digestion, were integrated into an "SCX-SAX" biphasic capillary column. To evaluate the performance of the developed microreactor, a mixture of bovine serum albumin, myoglobin, and cytochrome c was pretreated. Compared with the results obtained by the traditional in-solution process, the peptide recovery (93% vs 83%) and analysis throughput (3.5 vs 14 h) were obviously improved. The microreactor was further applied for the pretreatment of 14 μg of membrane proteins extracted from rat cerebellums, and 416 integral membrane proteins (IMPs) (43% of total protein groups) and 103 transmembrane peptides were identified by two-dimensional nanoliquid chromatography-electrospray ionization tandem mass spectrometry (2D nano-LC-ESI-MS/MS) in triplicate analysis. With the starting sample preparation amount decreased to as low as 50 ng, 64 IMPs and 17 transmembrane peptides were identified confidently, while those obtained by the traditional in-solution method were 10 and 1, respectively. All these results demonstrated that such an "SCX-SAX" based biphasic microreactor could offer a promising tool for the pretreatment of trace membrane proteins with high efficiency and throughput.
authors: National Chromatographic R. & A. Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science , Dalian 116023, China.
authors: Qun
authors: Q
authors: Zhao
authors: Yu
authors: Y
authors: Liang
authors: Huiming
authors: H
authors: Yuan
authors: Zhigang
authors: Z
authors: Sui
authors: Qi
authors: Q
authors: Wu
authors: Zhen
authors: Z
authors: Liang
authors: Lihua
authors: L
authors: Zhang
authors: Yukui
authors: Y
authors: Zhang
mesh_terms: D000478:Alkylation / Q000502:physiology; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D005561:Formates / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005561:Formates; D008565:Membrane Proteins; C030544:formic acid; D014357:Trypsin