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Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has attracted attention recently for large-scale top-down proteomics that aims to characterize proteoforms in cells at a global scale and with high throughput. In this work, CZE-MS/MS with ultraviolet photodissociation (UVPD) was evaluated for large-scale top-down proteomics for the first time. Roughly, 600 proteoforms and 369 proteins were identified from a zebrafish brain sample via coupling size exclusion chromatography (SEC) fractionation to CZE-UVPD. The dataset represents one of the largest top-down proteomics datasets using UVPD. Single-shot CZE-UVPD identified 227 proteoforms of 139 proteins from one SEC fraction of the zebrafish brain sample. The SEC-CZE-UVPD system identified zebrafish brain proteoforms in a mass range of 3-21 kDa. The UVPD with 213-nm photons produced reasonably good gas-phase fragmentation of proteoforms. For instance, 75% backbone cleavages were observed for Parvalbumin-7 with about 12-kDa molecular weight. The system detected various post-translational modifications (PTMs) from the zebrafish brain sample, including N-terminal acetylation, trimethylation and myristoylation of N-terminal glycine. Two different proteoforms of calmodulin, with either only N-terminal acetylation or both N-terminal acetylation and K115 trimethylation, were identified in the zebrafish brain sample. To our best knowledge, there is no experimental evidence reported in the literature on the two proteoforms of calmodulin in the zebrafish brain.

Capillary zone electrophoresis-tandem mass spectrometry with ultraviolet photodissociation (213 nm) for large-scale top–down proteomics