Comprehensive Online Two-Dimensional nanoLCxCZE-MS for Deep Top-Down Proteomics

Abstract

Abstract Understanding proteoforms, i.e., the various molecular forms in which proteins can exist, is important for deciphering biological processes and diseases. While capillary zone electrophoresis (CZE) proved advantageous for proteoform separation, limited sample loading capabilities restrict its application. Here, we present a novel comprehensive online coupling of nanoLC and CZE-MS (2D nanoLCxCZE-MS) for deep top-down proteomics (TDP). The 2D platform is highly automated, enabling robust performance and the possibility to perform proteoform quantitation as demonstrated by isobaric labeling experiments. The high orthogonality of reversed-phase LC and CZE results in a peak capacity of ∼2200, which leads to an increase in the number of identified proteoforms in a human Caucasian colon adenocarcinoma cell lysate sample by a factor of 3 compared to nanoLC-MS. Furthermore, CZE mobilities enable the attribution of many more proteoforms to a certain proteoform family at the MS1-level. Overall, the flexible platform enables highly efficient separation of intact proteoforms combined with sensitive MS-based TDP workflows, both for untargeted and targeted analysis of complex biological samples.