Modified Abstract. This research group recently published the first description of diagonal capillary electrophoresis. Diagonal capillary electrophoresis is a form of two-dimensional capillary electrophoresis that employs identical separation modes in each dimension. The distal end of the first capillary incorporates an enzyme-based microreactor. Analytes that are not modified by the reactor will have identical migration times in the two capillaries and will generate spots that fall on the diagonal in a reconstructed two-dimensional electropherogram. Analytes that undergo enzymatic modification in the reactor will have a different migration time in the second capillary and will generate spots that fall off the diagonal in the electropherogram. Dephosphorylation and desialylation result in the loss of a negative charge, which is trivial to detect using capillary electrophoresis. In this proposal, we will develop diagonal capillary electrophoresis as a robust tool for the determination of the phosphorylation status of complex samples. The technology offers significant advantages compared with alternatives. Most importantly, the technology provides accurate estimates of the extent of modification without artifact due to differences in ionization efficiency of the native and modified peptides. Also, the technology is fully automated: once the sample is loaded into the instrument, no fraction collection, pipetting, or other sample manipulations are performed. Finally, the technology employs capillary electrophoresis, which appears to be better suited than reversed-phase liquid chromatography for the analysis of peptides containing charged modifications.

Public Health Relevance

. The phosphorylation status of a protein modulates the activity of that protein, which is important in a wide range of diseases. This proposal provides the first fully automated method to identify phosphorylated peptides without interference.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM096767-03
Application #
8534195
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
2011-08-01
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
3
Fiscal Year
2013
Total Cost
$275,025
Indirect Cost
$91,675
Name
University of Notre Dame
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
Sun, Liangliang; Zhu, Guijie; Mou, Si et al. (2014) Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for quantitative parallel reaction monitoring of peptide abundance and single-shot proteomic analysis of a human cell line. J Chromatogr A 1359:303-8
Zhu, Guijie; Sun, Liangliang; Yan, Xiaojing et al. (2014) Bottom-up proteomics of Escherichia coli using dynamic pH junction preconcentration and capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry. Anal Chem 86:6331-6
Sun, Liangliang; Bertke, Michelle M; Champion, Matthew M et al. (2014) Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development. Sci Rep 4:4365
Zhu, Guijie; Sun, Liangliang; Yan, Xiaojing et al. (2014) Stable, reproducible, and automated capillary zone electrophoresis-tandem mass spectrometry system with an electrokinetically pumped sheath-flow nanospray interface. Anal Chim Acta 810:94-8
Huge, Bonnie Jaskowski; Flaherty, Ryan J; Dada, Oluwatosin O et al. (2014) Capillary electrophoresis coupled with automated fraction collection. Talanta 130:288-93
Sun, Liangliang; Zhu, Guijie; Yan, Xiaojing et al. (2014) Uncovering immobilized trypsin digestion features from large-scale proteome data generated by high-resolution mass spectrometry. J Chromatogr A 1337:40-7
Sun, Liangliang; Zhu, Guijie; Yan, Xiaojing et al. (2014) Capillary zone electrophoresis for analysis of complex proteomes using an electrokinetically pumped sheath flow nanospray interface. Proteomics 14:622-8
Sarver, Scott A; Chetwani, Nishant; Dovichi, Norman J et al. (2014) A comparison of alternating current and direct current electrospray ionization for mass spectrometry. J Am Soc Mass Spectrom 25:524-9
Zhao, Yimeng; Sun, Liangliang; Champion, Matthew M et al. (2014) Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry for top-down characterization of the Mycobacterium marinum secretome. Anal Chem 86:4873-8
Zhu, Guijie; Sun, Liangliang; Yan, Xiaojing et al. (2013) Single-shot proteomics using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry with production of more than 1250 Escherichia coli peptide identifications in a 50 min separation. Anal Chem 85:2569-73

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