The Potential of Polymer Monolithic Capillary Columns for the LC — MS Analysis of Intact Proteins.

LC-MS-based top-down proteomics involves the chromatographic separation and mass spectrometric characterization of intact proteins without prior enzymatic cleavage into peptides. This article describes the preparation of monolithic reversed-phase (RP) columns with optimized porous structure and their performance separating intact proteins. Peak capacities of around 600 could be achieved within a 2 h gradient. The combination of high-resolution monolith chromatography with high-accuracy time-of-flight mass spectrometrY (TOF-MS) allowed intact proteins and protein isoforms that differ only in their oxidation state to be distinguished. The article also describes various MS fragmentation techniques for the identification and the more detailed characterization of intact proteins. [ABSTRACT FROM AUTHOR]