Mass Spectrometric Analysis of Thiol Proteins/Peptides Following Selenamide Derivatization And Electrolytic Reduction of Disulfide Bonds

Ambient mass spectrometry (MS) methods such as desorption electrospray ionization-mass spectrometry (DESI-MS) provides direct ionization of analytes with little or no sample preparation. The capability for liquid samples analysis allows DESI-MS to be coupled with many devices such as electrochemical cells and liquid chromatography (LC) for novel analytical applications. The online coupling of a thin-layer electrochemical flow cell with liquid sample DESI-MS can be applied for investigating different electrochemical reactions of biological molecules such as electrolytic reduction of disulfide-containing peptides/proteins. In addition, electrochemistry (EC) can assist the top-down characterization of proteins by electrolytic reduction of the disulfide bonds, in which sequence coverage can be significantly improved for electron-capture dissociation (ECD) and collision-induced dissociation (CID) analysis. Furthermore, the online coupling of LC with liquid sample DESI-MS was established, which allows a wide range of elution flow rates, online derivatization via reactive DESI to solve the post-column derivatization problems, also integration with EC. In addition, reactive DESI, which exploits the potential for coupling specific ion/molecule reactions with the ionization event, greatly improves the selectivity and efficiency for saccharide detection.;A systemic mass spectrometric investigation of a novel strategy for labeling biological thiols using selenamide reagents, involving the cleavage of the Se-N bond and form a new Se-S bond was introduced. Among 20 natural amino acids, the reaction is highly selective to thiol-containing peptides and proteins and occurs rapidly in high yield. The derivatization is also reversible upon addition of reducting reagents. The MS/MS dissociation behaviors of resulting peptide ions upon CID and electron-transfer dissociation (ETD) were investigated. In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways, allowing fast screening of peptides/proteins containing free cysteine residues in a mixture. By contrast, ETD dissociation of the two selenamide-derivatized peptide ions show the facile loss of the tag, providing insight into the mechanism for electron-based ion dissociation. The derivatization reaction and related ion dissociation chemistry would find extensive applications in proteomics, including the derivatization of thiol protein/peptides arising from online electrolytic reduction mentioned above.