Capillary zone electrophoresis-tandem mass spectrometry with ultraviolet photodissociation (213 nm) for large-scale top–down proteomics†
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 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 with 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. UVPD with 213 nm photons produced reasonably good gas-phase fragmentation of proteoforms. For instance, 75% backbone cleavage was 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 the best of our knowledge, there is no experimental evidence reported in the literature on the two proteoforms of calmodulin in zebrafish brain.
- This article is part of the themed collection: Analytical Methods Emerging Investigators