title: Integration of electrochemistry with ultra-performance liquid chromatography/mass spectrometry.
abstract: This study presents the development of ultra-performance liquid chromatography (UPLC) mass spectrometry (MS) combined with electrochemistry (EC) for the first time and its application for the structural analysis of proteins/peptides that contain disulfide bonds. In our approach, a protein/peptide mixture sample undergoes a fast UPLC separation and subsequent electrochemical reduction in an electrochemical flow 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 disulfide bonds can be differentiated from those without disulfide bonds, as the former are electroactive and reducible. MS/MS analysis of the disulfide-reduced peptide ions provides increased information on the sequence and disulfide-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., α-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.
authors: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University, Athens, OH, 45701 USA. yc816211@ohio.edu.
authors: Yi
authors: Y
authors: Cai
authors: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University, Athens, OH, 45701 USA. qz320511@ohio.edu.
authors: Qiuling
authors: Q
authors: Zheng
authors: Department of Analytical Chemistry, Merck Research Laboratories, Merck & Co., Inc., Rahway, NJ 07065, USA. yong_liu2@merck.com.
authors: Yong
authors: Y
authors: Liu
authors: Department of Analytical Chemistry, Merck Research Laboratories, Merck & Co., Inc., Rahway, NJ 07065, USA. roy_helmy@merck.com.
authors: Roy
authors: R
authors: Helmy
authors: Dep artment of Chemistry and Biochemistry, Department of Biological Chemistry, David Geffen School of Medicine at UCLA, and UCLA/DOE Institute for Genomics and Proteomics, University of California-Los Angeles, Los Angeles, CA 90095, USA. roger.giles@srlab.co.uk.
authors: Joseph A
authors: JA
authors: Loo
authors: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University, Athens, OH, 45701 USA. chenh2@ohio.edu.
authors: Hao
authors: H
authors: Chen
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation*; D018511:Systems Integration
chemical_list: D011506:Proteins