Mechanistic Studies of Radical-Driven Peptide Tandem Mass Spectrometry: Implications for Tyrosine Sulfation Analysis and Higher Order Protein Structural Characterization
Jang, Eunju
2022
Abstract
Three-dimensional structure, established by the amino acid sequence, determines protein function. The amino acid sequence can be identified via tandem mass spectrometry (MS/MS) whereas hydrogen/deuterium exchange (HDX)-liquid chromatography/MS is widely used for determination of protein conformation. In this dissertation, mechanistic studies of radical-driven peptide MS/MS are presented for improved identification of the highly labile, biologically important posttranslational modification (PTM) tyrosine sulfation, and for enhanced structural resolution in HDX-MS. The generalizability of the alternative MS/MS method free radical initiated peptide sequencing (FRIPS) is explored in Chapter 2. We show that competition between desired radical-driven fragmentation pathways and undesired mobile proton-driven pathways appears dependent on the peptide charge state. Lower charge states promote radical-driven dissociation with high sequence coverage. Alternative positive ion mode charge carriers, including sodium and calcium ions, are shown to restore radical-driven fragmentation for peptides lacking basic amino acid residues. Tyrosine sulfation is particularly challenging to characterize in positive ion mode MS. In Chapter 3, the influence of basic amino acid residues and peptide chemical modifications on sulfopeptide stability are examined. We show that guanidination increases sulfopeptide stability during electrospray ionization and improves sulfate retention during MS/MS. We hypothesize that such groups may stabilize sulfate groups via salt bridge formation and/or by sequestering protons. Cysteine modifications also affect sulfopeptide stability. In particular, alkylation with fixed positive charge-containing vinyl pyridine resulted in several sulfate-retaining fragment ions following electron transfer dissociation (ETD). Such sulfated fragments allow direct sulfotyrosine identification within a peptide, a feat that has been elusive to date. Even more challenging is the determination of a sulfation site in peptides containing multiple tyrosine residues. In Chapter 4, the insights from Chapter 3 were applied towards sulfation site determination in a tyrosylprotein sulfotransferase 1 (TPST1) singly sulfated peptide containing three tyrosines. This peptide was previously detected in a proteomic analysis of rat liver Golgi; however, complete desulfation was observed upon higher energy collision dissociation (HCD) MS/MS. Both ETD and electron capture dissociation (ECD) allowed sulfation site determination in a synthetic, unmodified TPST1 tryptic peptide sulfated on the third tyrosine Guanidination resulted in the observation of sulfated fragment ions for the other two sulfopeptide isomers; however, lack of characteristic fragments precluded unambiguous identification of the sulfation site in these two sulfopeptides. By contrast, negative ion mode FRIPS was explored and enabled sulfation site determination in these two sulfopeptide isomers. Chapter 5 probes the degree of hydrogen/deuterium scrambling prior to ECD (which does not incur H/D scrambling) as a function of peptide size. “Soft” ion source/transfer conditions were previously shown to prevent scrambling for a 12-mer model peptide; however, such conditions typically significantly reduce ion abundance. We hypothesized that larger peptides may be more tolerant to typical ion source/transfer conditions, thus allowing wider implementation of ECD for enhancing structural resolution. Indeed we found that a shorter, 10-mer, peptide showed a higher degree of scrambling under the same “soft” conditions. By contrast, under “harsh” conditions where signal abundance is higher and the 12-mer peptide undergoes nearly compete scrambling, a longer, 16-mer, peptide showed moderate scrambling. Overall, the mechanistic studies and chemical derivatization strategies described in this dissertation will allow implementation of improved radical driven MS/MS methods to enable deeper insights into protein structure.Deep Blue DOI
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Radical-driven tandem mass spectrometry
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