Proteomics

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Automated capillary isoelectric focusing-tandem mass spectrometry for top-down proteomics

ABSTRACT: Mass spectrometry (MS)-based top-down proteomics (TDP) delineates proteoforms in cells and requires high-resolution proteoform separation. Herein, for the first time, we employed an automated capillary isoelectric focusing-tandem MS (cIEF-MS/MS) system for large-scale TDP of complex proteomes. Single-shot cIEF-MS/MS identified 771 proteoforms from an Escherichia coli (E. coli) proteome consuming only nanograms of proteins. Coupling two-dimensional size exclusion chromatography (SEC)-cIEF, named “gel-free 2D-PAGE”, to ESI-MS/MS enabled the identification of nearly 2000 proteoforms from the E. coli proteome. Label-free quantitative TDP of zebrafish male and female brains using the SEC-cIEF-MS/MS quantified thousands of proteoforms and revealed sex-dependent proteoform profiles in brains. We discovered several proteolytic proteoforms of pro-opiomelanocortin and prodynorphin with significantly higher abundance in male brains as potential endogenous hormone proteoforms. Multi-level quantitative proteomic analyses (TDP and bottom-up proteomics (BUP)) of the brains revealed that majority of proteoforms having statistically significant difference in abundance between genders (TDP) showed no abundance difference at the corresponding protein group level (BUP), which highlights the importance of delineating proteins in a proteoform-specific manner for accurately understanding protein function.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Escherichia Coli Danio Rerio (zebrafish) (brachydanio Rerio)

TISSUE(S): Brain

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD020342 | Pride | 2020-12-08

REPOSITORIES: Pride

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Publications

Automated Capillary Isoelectric Focusing-Tandem Mass Spectrometry for Qualitative and Quantitative Top-Down Proteomics.

Xu Tian T   Shen Xiaojing X   Yang Zhichang Z   Chen Daoyang D   Lubeckyj Rachele A RA   McCool Elijah N EN   Sun Liangliang L  

Analytical chemistry 20201202 24

Top-down proteomics (TDP) aims to delineate proteomes in a proteoform-specific manner, which is vital for accurately understanding protein function in cellular processes. It requires high-capacity separation of proteoforms before mass spectrometry (MS) and tandem MS (MS/MS). Capillary isoelectric focusing (cIEF)-MS has been recognized as a useful tool for TDP in the 1990s because cIEF is capable of high-resolution separation of proteoforms. Previous cIEF-MS studies concentrated on measuring the  ...[more]

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