Multiplexed Mass Spectrometric Approaches for Protein Cross-Linking Analysis and Protein Functional Studies.

Abstract

Mass spectrometry(MS) alone, or combined with chemical cross-linking and proteolysis, is a valuable method in protein characterization. However, detection of cross-linked peptides remains challenging due to ion suppression and cross-linked peptide signals are difficult to differentiate from those of unmodified peptides. In this thesis, efforts to design a novel chemical cross-linker with metal oxide enrichment potential or unique gas-phase fragmentation pattern are presented. A series of biological studies, including mapping of protein-peptide interactions in the transcriptional machinery and determination of enzyme function and functional differentiation in natural product biosynthesis, were also performed. Photo-cross-linking combined with multiplexed MS and biotin-avidin enrichment was employed to map binding sites between gene transcriptional activators and a co-activator(mediator) with resolution up to one amino acid residue. Unprecedented C-C and C-S bond cleavages were observed in matrix-assisted laser desorption/ionization tandem time-of-flight collision induced dissociation(MALDI TOF/TOF CID) of photo-cross-linked products. A novel phosphate-containing cross-linker was synthesized and demonstrated to have amine reactivity. The corresponding cross-linked product was selectively enriched by metal dioxides.Gas-phase fragmentation patterns of peptides linked with disulfide-containing cross-linkers were examined in electron capture dissociation(ECD), electron detachment dissociation(EDD), negative ion CID, and MALDI TOF/TOF CID, to find an optimal gas-phase cleavage method for identification of such cross-linked peptides.Iodinated peptides were subjected to ECD and electron transfer dissociation(ETD), showing highly selective C-I bond cleavage.A proximate, either through bond or through space, positive charge appears to facilitate C-I bond cleavage.These results provide a guide to design novel gas-phase cleavable cross-linkers and an additional model for elucidating the ECD/ETD mechanism. Quadrupole fractionation was also applied in this thesis to enhance detection in the 350-600 m/z region. Fourier transform ion cyclotron resonance(FT-ICR) MS and infrared multiphoton dissociation(IRMPD) were applied to characterize the function and functional differentiation of enzymes from natural product biosynthetic pathways. GCN5-related N-acetyltransferase and enoyl reductase(ER) involved in Curacin A(Cur) biosynthesis were shown to have the unprecedented functions of dual decarboxylation/thiol acetyl transfer and cyclopropanation, respectively. Catalytic efficiencies of Cur and Jamaicamide ERs and site-specific mutants of Cur halogenase were measured by IRMPD. The role of a sulfotransferase in decarboxylative chain termination in Cur biosynthesis was also elucidated.