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Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast.
- Source :
-
Journal of proteome research [J Proteome Res] 2016 Oct 07; Vol. 15 (10), pp. 3679-3685. Date of Electronic Publication: 2016 Aug 25. - Publication Year :
- 2016
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Abstract
- We used reversed-phase liquid chromatography to separate the yeast proteome into 23 fractions. These fractions were then analyzed using capillary zone electrophoresis (CZE) coupled to a Q-Exactive HF mass spectrometer using an electrokinetically pumped sheath flow interface. The parameters of the mass spectrometer were first optimized for top-down proteomics using a mixture of seven model proteins; we observed that intact protein mode with a trapping pressure of 0.2 and normalized collision energy of 20% produced the highest intact protein signals and most protein identifications. Then, we applied the optimized parameters for analysis of the fractionated yeast proteome. From this, 580 proteoforms and 180 protein groups were identified via database searching of the MS/MS spectra. This number of proteoform identifications is two times larger than that of previous CZE-MS/MS studies. An additional 3,243 protein species were detected based on the parent ion spectra. Post-translational modifications including N-terminal acetylation, signal peptide removal, and oxidation were identified.
Details
- Language :
- English
- ISSN :
- 1535-3907
- Volume :
- 15
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- Journal of proteome research
- Publication Type :
- Academic Journal
- Accession number :
- 27490796
- Full Text :
- https://doi.org/10.1021/acs.jproteome.6b00493