Strong cation exchange-reversed phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry platform with high peak capacity for deep bottom-up proteomics

Chen, Daoyang; Shen, Xiaojing; Sun, Liangliang

doi:10.1016/j.aca.2018.01.037

Cited by 47Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsMicrofluidic and Capillary Electrophoresis Applications

Strong cation exchange-reversed phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry platform with high peak capacity for deep bottom-up proteomics

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Chen, Daoyang, Shen, Xiaojing, Sun, Liangliang·2018·Analytica Chimica Acta

DOI 10.1016/j.aca.2018.01.037PMC PMC5831384

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Abstract

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 8000 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 8200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 h. 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 7000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8200 vs. 8900 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.

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Capillary zone electrophoresis–mass spectrometry with microliter-scale loading capacity, 140 min separation window and high peak capacity for bottom-up proteomics
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Self-citation

Authors: Chen, D., Shen, X., & Sun, L.
Affiliations: Michigan State University (US)
Corresponding author: Sun, Liangliang
ORCID: Chen, Daoyang
Shen, Xiaojing
Sun, Liangliang
Venue: Analytica Chimica Acta
Publisher: Elsevier BV
Published: 2018-07
Volume: 1012
Pages: 1-9
ISSN: 0003-2670 (ISSN-L)
DOI: 10.1016/j.aca.2018.01.037
PMC: PMC5831384
OpenAlex: W2791606096
Cited by: 47
References: 39
Citations by year: 2026: 1 · 2025: 2 · 2024: 1 · 2023: 4 · 2022: 3 · 2021: 8 · 2020: 8 · 2019: 8 · 2018: 11 · 2016: 1
FWCI: 3.78
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: www.elsevier.com/tdm/userlicense/1.0/ (tdm)
Locations: Analytica Chimica Acta (publishedVersion)
PubMed (publishedVersion)
Europe PMC (PubMed Central) (acceptedVersion)
PubMed Central (acceptedVersion)
Funders: Michigan State University
National Institute of General Medical Sciences, National Institutes of Health (R01GM125991)
Topics: Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
Keywords: strong cation exchange, capillary zone electrophoresis, reversed-phase liquid chromatography, shotgun proteomics, peak capacity, protein identification, peptide identification
MeSH terms: Polyacrylamides, Acrylic Resins, Animals, Brain, Chromatography, High Pressure Liquid, Hydrogen-Ion Concentration, Electrophoresis, Capillary, Proteomics, Mice, Tandem Mass Spectrometry, Chromatography, Reverse-Phase

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Strong cation exchange-reversed phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry platform with high peak capacity for deep bottom-up proteomics

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