Development of Polymer-Based Hydrophobic Monolithic Columns and Their Applications in Proteome Analysis

Abstract

As described in Chap. 2, the LC separation capability is one of the most important factors to achieve high proteome coverage in shotgun proteome analysis. In order to improve the separation efficiency, long columns packed with smaller particles are commonly applied. Though the operating pressure of the ultra-high performance liquid chromatography instrument is often over 10,000 psi, the packed length of separation column is still limited, especially when capillary columns with smaller inner diameters (<50 μm) are utilized in nanoflow liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Further, the column packing is difficult and time-consuming when column with smaller dimension and smaller packing material (< 5 μm) is utilized. Monolithic columns have higher permeability and faster mass transferring rate than columns packed with particle materials due to its porous structure, which makes them good alternatives to packed columns in chromatography separation. Especially in shotgun proteome analysis, where capillary column (usually 10–100 μm i.d.) is adopted to increase the sensitivity as well as extremely complex protein samples such as serum is inevitably confronted, monolithic capillary columns are feasibly explored to improve the LC separation performance.