Abstract
This study presents the development of ultra-performance liquid chromatography (UPLC) mass spectrometry (MS) combined with electrochemistry (EC) for the frst time and its application for the structural analysis of proteins/peptides that contain disulfde bonds. In our approach, a protein/peptide mixture sample undergoes a fast UPLC separation and subsequent electrochemical reduction in an electrochemical fow cell followed by online MS and tandem mass spectrometry (MS/MS) analyses. The electrochemical cell is coupled to the mass spectrometer using our recently developed desorption electrospray ionization (DESI) interface. Using this UPLC/EC/DESI-MS method, peptides that contain disulfde bonds can be differentiated from those without disulfde bonds, as the former are electroactive and reducible. MS/MS analysis of the disulfde-reduced peptide ions provides increased information on the sequence and disulfde-linkage pattern. In a reactive DESI-MS detection experiment in which a supercharging reagent was used to dope the DESI spray solvent, increased charging was obtained for the UPLC-separated proteins. Strikingly, upon online electrolytic reduction, supercharged proteins (e.g., a-lactalbumin) showed even higher charging, which will be useful in top-down protein structure MS analysis as increased charges are known to promote protein ion dissociation. Also, the separation speed and sensitivity are enhanced by approximately 1~2 orders of magnitude by using UPLC for the liquid chromatography (LC)/EC/MS platform, in comparison to the previously used high-performance liquid chromatography (HPLC). This UPLC/EC/DESI-MS method combines the power of fast UPLC separation, fast electrochemical conversion, and online MS structural analysis for a potentially valuable tool for proteomics research and bioanalysis.
Original language | English (US) |
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Pages (from-to) | 341-351 |
Number of pages | 11 |
Journal | European Journal of Mass Spectrometry |
Volume | 21 |
Issue number | 3 |
DOIs | |
State | Published - Mar 13 2015 |
Externally published | Yes |
All Science Journal Classification (ASJC) codes
- Atomic and Molecular Physics, and Optics
- Spectroscopy
Keywords
- DESI-MS
- Disulfde-bond reduction
- Electrochemistry
- Protein
- Supercharging
- UPLC