Capillary Trap Column with Strong Cation-Exchange Monolith for Automated Shotgun Proteome Analysis

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Abstract
A 150 μm internal diameter capillary monolithic column with a strong cation-exchange stationary phase was prepared by direct in situ polymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent consisting dimethylsulfoxide, dodecanol, and N,N‘-dimethylformamide. This phosphate monolithic column exhibits higher dynamic binding capacity, faster kinetic adsorption of peptides, and more than 10 times higher permeability than the column packed with commercially available strong cation-exchange particles. It was applied as a trap column in a nanoflow liquid chromatography−tandem mass spectrometry system for automated sample injection and online multidimensional separation. It was observed that the sample could be loaded at a flow rate as high as 40 μL/min with a back pressure of ∼1300 psi and without compromising the separation efficiency. Because of its good orthogonality to the reversed phase separation mechanism, the phosphate monolithic trap column was coupled with a reversed-phase column for online multidimensional separation of 19 μg of the tryptic digest of yeast proteins. A total of 1522 distinct proteins were identified from 5608 unique peptides (total of 54 780 peptides) at the false positive rate only 0.46%.