Dataset Information

Capillary zone electrophoresis-tandem mass spectrometry as an alternative to liquid chromatography-tandem mass spectrometry for top-down proteomics of histones with the identification of over 1000 histone proteoforms

ABSTRACT: Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified 80% more proteoforms than RPLC-MS/MS (589 vs. 322) from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. 3 µg), indicating its substantially higher sensitivity. The electrophoretic mobility (µef) of unmodified histone proteoforms can be predicted accurately (R2=0.98) with an optimized semi-empirical model based on our recent work, and we revealed that N-terminal acetylation had minimal effect on histone proteoforms’ µef. We identified 1108 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We achieved delineation of histone proteoforms regarding post-translational modifications (PTMs) and their combinations, relative abundance among different proteoforms of the same protein, as well as the frequency of PTM occurrence on histone proteoforms. We identified histone proteoforms carrying various PTMs, e.g., acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. We revealed that different histone proteins had drastically different patterns of PTMs. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Bos Taurus (bovine)

SUBMITTER: Liangliang Sun

LAB HEAD: Liangliang Sun

PROVIDER: PXD021889 | Pride | 2022-04-04

REPOSITORIES: Pride

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Publications

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones.

Chen Daoyang D Yang Zhichang Z Shen Xiaojing X Sun Liangliang L

Analytical chemistry 20210302 10

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-fl ...[more]

PMID: 33650845

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Project description:Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep bottom-up proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8,000 protein identifications (IDs) from mammalian cell lines or tissues in 1-3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for bottom-up proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep bottom-up proteomics. The platform identified around 8,200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 hours. The data represents the largest bottom-up proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7,000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8,200 vs. 8,900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.

Dataset Information

Capillary zone electrophoresis-tandem mass spectrometry as an alternative to liquid chromatography-tandem mass spectrometry for top-down proteomics of histones with the identification of over 1000 histone proteoforms

Publications

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones.

Similar Datasets

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