Improving the Comprehensiveness and Sensitivity of Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry for Proteomic Analysis
Open accessAbstract
We describe a solid phase microextraction (SPME), multistep elution, transient isotachophoresis (tITP) capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) procedure which employs a high sensitivity porous electrospray ionization (ESI) sprayer for the proteomic analysis of a moderately complex protein mixture. In order to improve comprehensiveness and sensitivity over a previously reported proteomic application of the ESI sprayer, we evaluated preconcentration with SPME and multistep elution prior to tITP stacking and CE separation. To maximize separation efficiency, we primarily employed electrokinetic methods for elution and separation after loading the sample by application of pressure. Conditions were developed for optimum simultaneous electrokinetic elution and sample stacking using a tryptic digest of 16 proteins to maximize peptide identifications and minimize band broadening. We performed comparative proteomic analysis of a dilution series using CE and nanoflow liquid chromatography (nLC). We found complementary peptide and protein identifications with larger quantities (100 ng) of a Pyrococcus furiosus tryptic digest, but with mass-limited amounts (5 ng) CE was 3 times more effective at identifying proteins. We attribute these gains in sensitivity to lower noise levels with the porous CE sprayer, illustrated by better signal-to-noise ratios of peptide precursor ions and associated higher XCorr values of identified peptides when compared directly to nLC. From comparative analysis of SPME-tITP-CE with direct injection CE, the SPME-tITP process improved comprehensiveness and sensitivity.
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Many studies have suggested that CZE separations are more capable of identifying hydrophilic peptides than LC.
“Many studies have suggested that CZE separations are more capable of identifying hydrophilic peptides than LC48,87.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [87]
A polyacrylamide neutral coated capillary method achieved high reproducibility, with relative standard deviation for migration time in the separation window below 1%.
“These studies showed that high reproducibility with a relative standard deviation for migration time in separation window lower than 1% was achieved.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [108]
Reversed-phase SPE combined with tITP for a Pyrococcus furiosus tryptic digest gave low background noise and high sensitivity.
“Wang et al. used reversed-phase SPE in conjunction with tITP for sample concentration of a Pyrococcus furiosus tryptic digest, which resulted in low background noise and high sensitivity87.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [87]
Sample loading is the major drawback of CE and can be addressed using simple protocols, as shown in top-down and bottom-up applications.
“Sample loading is the major drawback of CE, which can be addressed using simple protocols, as demonstrated in top-down and bottom-up applications52,87.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [87]
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- Authors
- Wang, Y., Fonslow, B. R., Wong, C. C. L., Nakorchevsky, A., & Yates, J. R.
- Affiliations
- Scripps Research Institute (US)
- Corresponding author
- Yates, John R.
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2012-10-05
- Volume
- 84
- Issue
- 20
- Pages
- 8505-8513
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC3498465
- OpenAlex
- W2055979718
- Cited by
- 144
- References
- 46
- Citations by year
- 2026: 4 · 2025: 7 · 2024: 7 · 2023: 3 · 2022: 4 · 2021: 9 · 2020: 6 · 2019: 8 · 2018: 11 · 2017: 16 · 2016: 17 · 2015: 16 · 2014: 17 · 2013: 19
- FWCI
- 9.47
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health
- National Heart, Lung, and Blood Institute (HV-10-5, HHSN268201000035C)
- National Institute of Mental Health (R01MH067880)
- National Institute of General Medical Sciences (P41GM103533)
- National Institute of Allergy and Infectious Diseases (U19AI063603)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK074798)
- National Center for Research Resources (P41RR011823)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- tandem mass spectrometry, solid-phase microextraction, transient isotachophoresis, proteomics, preconcentration, protein identification, Pyrococcus furiosus, signal-to-noise ratio
- MeSH terms
- Animals, Escherichia coli, Proteins, Sensitivity and Specificity, Electrophoresis, Capillary, Archaeal Proteins, Pyrococcus furiosus, Escherichia coli Proteins, Proteomics, Solid Phase Microextraction, Tandem Mass Spectrometry, Isotachophoresis
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