Cited by 57Advanced Proteomics Techniques and ApplicationsMass Spectrometry Techniques and ApplicationsMicrofluidic and Capillary Electrophoresis Applications

Quantitative Proteomics Using Ultralow Flow Capillary Electrophoresis–Mass Spectrometry

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Faserl, Klaus, Kremser, Leopold, Müller, Martin, Teis, David, Lindner, Herbert H.·2015·Analytical Chemistry

Abstract

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE–MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE–MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE–MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE–MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE–MS analysis.

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  1. Lindner H.; Sarg B.; Helliger W. J. Capillary Electrophor. Microchip Technol. 2003, 8, 59– 67. 14596337
  2. Smith R. D.; Barinaga C. J.; Udseth H. R. Anal. Chem. 1988, 60, 1948– 1952.
  3. Lee E. D.; Muck W.; Henion J. D.; Covey T. R. Biomed. Environ. Mass Spectrom. 1989, 18, 844– 850.
  4. Olivares J. A.; Nguyen N. T.; Yonker C. R.; Smith R. D. Anal. Chem. 1987, 59, 1230– 1232.

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Authors
Faserl, K., Kremser, L., Müller, M., Teis, D., & Lindner, H. H.
Affiliations
  • Innsbruck Medical University (AT)
  • Innsbruck Medical University , , Innrain 80-82 , ,
Corresponding author
Lindner, Herbert H.
Venue
Analytical Chemistry
Published
2015-04-03
Volume
87
Issue
9
Pages
4633-4640
ISSN
  • 0003-2700 (ISSN-L)
  • 1520-6882
OpenAlex
W1985017503
Cited by
57
References
46
Citations by year
2026: 2 · 2024: 1 · 2023: 2 · 2022: 3 · 2021: 2 · 2020: 2 · 2019: 7 · 2018: 13 · 2017: 11 · 2016: 9 · 2015: 4 · 2013: 1
FWCI
5.49
Type
journal article
Language
en
Open access
Locations
  • Analytical Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
Austrian Science Fund (Y444-B12)
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
quantitative proteomics, capillary electrophoresis-mass spectrometry, yeast proteome, phosphopeptides, modified peptides, peptide fractionation, reversed-phase HPLC, protein quantification, differential regulation
MeSH terms
Chromatography, Liquid, Peptides, Proteins, Mass Spectrometry, Electrophoresis, Capillary, Proteome, Proteomics

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