Bottom-Up Proteomics of Escherichia coli Using Dynamic pH Junction Preconcentration and Capillary Zone Electrophoresis-Electrospray Ionization-Tandem Mass Spectrometry
Open accessAbstract
We report the use of the dynamic pH junction based capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) for bottom-up proteomics with an electrokinetically pumped sheath-flow nanospray capillary electrophoresis-mass spectrometry (CE-MS) interface and both LTQ-XL and LTQ-Orbitrap-Velos mass spectrometers. Conventional injection of 20 nL of a 1 mg/mL BSA digest identified 37 peptides and produced 66% sequence coverage. In contrast, pH junction injection of 130 nL (or larger) of a 0.05 mg/mL BSA digest identified 40 peptides and produced 70% coverage using a pH 6.5 sample buffer and the LTQ. A 20 nL conventional injection of a 1 mg/mL Escherichia coli digest identified 508 peptides and 199 proteins with the LTQ. A 400 nL pH junction injection of a 0.1 mg/mL E. coli digest identified 527 peptides and 179 proteins with the LTQ. Triplicate technical replicates of a 0.01 mg/mL sample with 400-nL injection volume using a pH junction identified 288 ± 9 peptides and 121 ± 5 proteins with the LTQ. There was outstanding concordance in migration time between the pH junction and normal injection. The pH junction produced narrower peaks and significant concentration for all but the most acidic components in the sample. Compared with the conventional stacking method, the pH junction method can generate comparable performance for small injection volume (20 nL) and significantly better concentration performance for a large injection volume (200 nL). We also applied the pH junction to three intact standard proteins and observed a >10× increase in peak intensity compared to conventional injection.
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A 130 nL pH junction injection of BSA identified 40 peptides and produced 70% coverage.
“In this study, a pH junction injection of 130 nL of BSA resulted in the identification of 40 peptides and produced 70% coverage.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [119]
Reproducibility of migration times between pH junction and normal injection was good.
“The authors found that reproducibility of the migration times between the pH junction and normal injection was good.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [119]
A 400 nL pH junction injection of an E. coli sample identified 527 peptides and 179 proteins.
“A 400 nL pH junction injection of an Escherichia coli sample enabled the identification of 527 peptides and 179 proteins.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [119]
Extending pH junction to three intact standard proteins increased peak intensity more than 10-fold compared to conventional injection.
“The pH junction was further extended to three intact standard proteins where the peak intensity increased more than 10-fold compared to conventional injection.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [119]
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- Authors
- Zhu, G., Sun, L., Yan, X., & Dovichi, N. J.
- Affiliations
- University of Notre Dame (US)
- University of Notre Dame , , , ,
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2014-05-22
- Volume
- 86
- Issue
- 13
- Pages
- 6331-6336
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC4082393
- OpenAlex
- W2040282929
- Cited by
- 78
- References
- 22
- Citations by year
- 2026: 5 · 2025: 1 · 2024: 2 · 2023: 4 · 2022: 2 · 2021: 4 · 2020: 5 · 2019: 6 · 2018: 7 · 2017: 7 · 2016: 12 · 2015: 18 · 2014: 4 · 2013: 1
- FWCI
- 5.86
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Analytical Chemistry (publishedVersion, other-oa)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Institute of General Medical Sciences (R01GM096767)
- 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
- shotgun proteomics, Escherichia coli, dynamic pH junction, capillary zone electrophoresis, preconcentration, peptide sequence coverage, protein identification
- MeSH terms
- Animals, Cattle, Equipment Design, Escherichia coli, Hydrogen-Ion Concentration, Serum Albumin, Bovine, Electrophoresis, Capillary, Spectrometry, Mass, Electrospray Ionization, Escherichia coli Proteins, Proteomics, Tandem Mass Spectrometry
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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