Online Multidimensional Separation with Biphasic Monolithic Capillary Column for Shotgun Proteome Analysis Fangjun Wang, Jing Dong, Mingliang Ye, Xiaogang Jiang, Ren’an Wu, and Hanfa Zou* National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Received August 27, 2007 Abstract: A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 μm i.d. capillary. Separation perfor- mance of this column was evaluated by a five-cycle online multidimensional separation of 10 μg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only ∼900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely at- tributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation. Keywords: Biphasic monolithic column • shotgun prote- ome analysis • multidimensional separation • μHPLC-MS/MS Introduction Multidimensional separation is the most powerful approach to increase the separation performance in liquid chromatog- raphy (LC) analysis, and the separation peak capacity could be improved greatly by combining two dimensions of separa- tions with orthogonal mechanisms. 1,2 Though off-line multi- dimensional separation can provide more sufficient prefrac- tionation for analytes, 3,4 online multidimensional separation displays advantages such as higher sensitivity, minimal loss of sample, no vial contamination, and no sample dilution effect. 5–8 Especially in capillary liquid chromatography-tandem mass spectrometry (μHPLC-MS/MS), online multidimensional sepa- ration exhibits one of the best solutions for analysis of the limited amount of biological samples. Multidimensional protein identification technology (MudPIT) developed by Yates and co- workers has been successfully applied in shotgun proteome analysis for its prominent separation performance and good compatibility to mass spectrometry (MS) detector. 9–11 In the classical mode of this technology, strong-cation exchange (SCX) and reversed-phase (RP) particles are packed in sequence within a single capillary, and peptides binding initially onto the SCX segment are fractionated with stepwise elution of salt buffer followed with gradient separation on the RP segment before MS detection. However, as a packed column is used, the packing length of the both segments is limited by the operating pressure of the LC system. Therefore, the sample loading capacity, as well as separation capability of this type of biphasic packed column, is hard to increase unless ultrahigh pressure system is used. 12 Monolithic columns with polymeric stationary phases are widely applied in LC analysis due to their advantages such as pH stability, good permeability, inertness to biomolecules, absence of deleterious effects from silanol, and facility for modification. 13–15 Recently, Jmeian et al. applied tandem affinity monolithic microcolumns to deplete the high-abun- dance proteins in serum samples, and zero dead volume (ZDV) union was used to connect the monolithic columns in se- quence. 16 We have prepared a SCX monolithic column based on the functional monomer ethylene glycol methacrylate phosphate (EGMP) 17 to trap and fractionate peptides in multidimensional separation, and this type of phosphate monolithic column has higher dynamic binding capacity than column packed with commercially available Polysulfethyl A. A neutrally hydrophobic monolithic column based on the func- tional monomer lauryl methacrylate (LMA) was also prepared for capillary electrochromatography (CEC) separation of ionic compounds driven by electrophoretic mobility. 18 It can be expected that these two types of monolithic columns should have good orthogonality in retention mechanism for multidi- mensional separation. Therefore, after optimizing the poly- merization mixture constitution for preparation of the LMA- based monolith, a biphasic monolithic column was synthesized with 10 cm segment of SCX monolith and 65 cm segment of RP monolith in a single 100 μm i.d. capillary in this study. This biphasic monolithic column was applied in online multidimen- sional separation for shotgun proteome analysis under relatively low operating pressure of ∼900 psi, and good separation perfor- mance of this biphasic column was demonstrated. Experimental Section Materials. Fused silica capillaries were purchased from Yongnian Optical Fiber Factory (Hebei, China). Azobisisobu- tyronitrile (AIBN) was obtained from Shanghai Fourth Reagent Plant (Shanghai, China). Other materials were purchased from Sigma (St. Louis, MO). Preparation of the Tryptic Digest of Yeast Proteins. The yeast protein extract was prepared in a denaturing buffer * Correspondence: Prof. Dr. Hanfa Zou, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Tel: +86-411-84379610. Fax: +86-411-84379620. E-mail: hanfazou@dicp.ac.cn. 306 Journal of Proteome Research 2008, 7, 306–310 10.1021/pr700562b CCC: $40.75  2008 American Chemical Society Published on Web 12/08/2007