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

ABSTRACT: 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.

SUBMITTER: McCool EN

PROVIDER: S-EPMC6788745 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

McCool Elijah N EN Chen Daoyang D Li Wenxue W Liu Yansheng Y Sun Liangliang L

Analytical methods : advancing methods and applications 20190507 22

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) ...[more]

PMID: 31608127

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Project description:Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.

Dataset Information

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

Publications

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

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