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 and sialic acid 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 and glycosylation 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 and specific glycosylated peptides without interference.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM096767-01
Application #
8024455
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
2011-08-01
Budget End
2012-07-31
Support Year
1
Fiscal Year
2011
Total Cost
$285,000
Indirect Cost
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
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Zhang, Zhenbin; Peuchen, Elizabeth H; Dovichi, Norman J (2017) Surface-Confined Aqueous Reversible Addition-Fragmentation Chain Transfer (SCARAFT) Polymerization Method for Preparation of Coated Capillary Leads to over 10 000 Peptides Identified from 25 ng HeLa Digest by Using Capillary Zone Electrophoresis-Tandem Ma Anal Chem 89:6774-6780
Boley, Danielle A; Zhang, Zhenbin; Dovichi, Norman J (2017) Multisegment injections improve peptide identification rates in capillary zone electrophoresis-based bottom-up proteomics. J Chromatogr A 1523:123-126
Sun, Liangliang; Dubiak, Kyle M; Peuchen, Elizabeth H et al. (2016) Single Cell Proteomics Using Frog (Xenopus laevis) Blastomeres Isolated from Early Stage Embryos, Which Form a Geometric Progression in Protein Content. Anal Chem 88:6653-7
Zhu, Guijie; Sun, Liangliang; Dovichi, Norman J (2016) Thermally-initiated free radical polymerization for reproducible production of stable linear polyacrylamide coated capillaries, and their application to proteomic analysis using capillary zone electrophoresis-mass spectrometry. Talanta 146:839-43

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