title: In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry.
abstract: Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S-S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the "parent" disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.
authors.affiliation: Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands. l.switzar@lumc.nl.|Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Leiden, The Netherlands. l.switzar@lumc.nl.|, Albinusdreef 2, Postzone S3, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. l.switzar@lumc.nl.
authors.forename: Linda
authors.initials: L
authors.lastname: Switzar
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Simone
authors.initials: S
authors.lastname: Nicolardi
authors.affiliation: Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.|Department of Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Julie W
authors.initials: JW
authors.lastname: Rutten
authors.affiliation: Department of Clinical Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Saskia A J Lesnik
authors.initials: SA
authors.lastname: Oberstein
authors.affiliation: Department of Human Genetics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Annemieke
authors.initials: A
authors.lastname: Aartsma-Rus
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
authors.forename: Yuri E M
authors.initials: YE
authors.lastname: van der Burgt
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D007782:Lactoglobulins; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D012260:Ribonucleases
chemical_list: D004220:Disulfides; D007782:Lactoglobulins; D010446:Peptide Fragments; D012260:Ribonucleases; C029600:ribonuclease B
keywords: Electrochemistry; High resolution mass spectrometry; Protein disulfide bonds