Accurate Determination of Peptide Phosphorylation Stoichiometry Via Automated Diagonal Capillary Electrophoresis Coupled with Mass Spectrometry: Proof of Principle
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While reversible protein phosphorylation plays an important role in many cellular processes, simple and reliable measurement of the stoichiometry of phosphorylation can be challenging. This measurement is confounded by differences in the ionization efficiency of phosphorylated and unphosphorylated sites during analysis by mass spectrometry. Here, we demonstrate diagonal capillary electrophoresis-mass spectrometry for the accurate determination of this stoichiometry. Diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized alkaline phosphatase microreactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. The first dimension is used to separate a mixture of the phosphorylated and unphosphorylated forms of a peptide. Fractions are parked in the reactor where they undergo complete dephosphorylation. The products are then periodically transferred to the second capillary and analyzed by mass spectrometry (MS). Because the phosphorylated and unphosphorylated forms differ in charge, they are well resolved in the first dimension separation. Because the unphosphorylated and dephosphorylated peptides are identical, there is no bias in ionization efficiency, and phosphorylation stoichiometry can be determined by the ratio of the signal of the two forms. A calibration curve was generated from mixtures of a phosphorylated standard peptide and its unphosphorylated form, prepared in a bovine serum albumin tryptic digest. This proof of principle experiment demonstrated a linear response across nearly 2 orders of magnitude in stoichiometry.
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Post-translational phosphorylation plays critical roles in biological systems and can influence the electrophoretic mobility of peptides.
“Post-translational phosphorylation plays critical roles in biological systems83 and it can also influence the electrophoretic mobility of peptides.”
From Recent trends of capillary electrophoresis‐mass spectrometry in proteomics research (2019) · cites this paper as [83]
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- Authors
- Mou, S., Sun, L., & Dovichi, N. J.
- Affiliations
- University of Notre Dame (US)
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2013-11-05
- Volume
- 85
- Issue
- 22
- Pages
- 10692-10696
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- PMC
- PMC3873722
- OpenAlex
- W1965762619
- Cited by
- 22
- References
- 32
- Citations by year
- 2025: 1 · 2022: 1 · 2021: 1 · 2019: 2 · 2018: 5 · 2016: 2 · 2015: 3 · 2014: 6 · 2013: 1
- FWCI
- 1.80
- 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 institute of Health and Medical Research (R01GM096767)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Microfluidic and Capillary Electrophoresis Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Keywords
- peptide phosphorylation, phosphorylation stoichiometry, dephosphorylation, two-dimensional separation, protein phosphorylation, calibration curve, bovine serum albumin
- MeSH terms
- Alkaline Phosphatase, Animals, Cattle, Peptide Fragments, Phosphopeptides, Phosphorylation, Receptor, Insulin, Serum Albumin, Bovine, Trypsin, Electrophoresis, Capillary, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry
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