Optimization of Acidic Post-translational Modification Detection and Natural Product Biosynthetic Enzyme Analysis with Dual Polarity nLC/FT-ICR Mass Spectrometry

Mass spectrometry-based proteomic protocols can identify thousands of expressed proteins with widely varying concentrations. However, acidic post-translational modifications (PTMs), e.g., phosphorylation and sulfation, are difficult to examine, due to the reduced ionization efficiency of highly acidic peptides with positive ion mode nanoelectrospray ionization (nESI). This thesis presents methods for improved detection of acidic, modified peptides and natural product biosynthetic active site peptides in both positive and negative ion mode. Trace addition of trifluoroethanol (TFE) to aqueous samples suppresses corona discharge typically observed in negative ion mode nESI experiments. TFE greatly (~8 fold) improves nESI spray stability without altering observed protein, peptide, and small molecule charge states. This phenomenon is likely due to the highly electronegative fluorine atom’s ability to scavenge electrons, thus stemming plasma formation. In negative ion mode nanoflow liquid chromatography-mass spectrometry (nLC-MS) experiments, TFE addition increases the number of identified peptides by 18%. The relatively simple addition of TFE to sample solutions for improved negative ion mode nESI can be readily employed for improved analysis of widely varied compounds in direct infusion and nLC-MS experiments. The demonstrated compatibility of TFE with nLC-MS allowed for systematic examination of mobile phase and detection polarity effects on peptide identifications. Regardless of mobile phase pH and detection polarity, overall sequence coverage for a six protein digest was similar. However, multiply phosphorylated peptides were only detected at pH 11 in negative ion mode and sulfopeptides were detected most effectively (~55 fold improvement) in negative ion mode and with maximum ion abundance at pH 11. This work demonstrates that alkaline pH separations coupled with negative ion mode nESI provides an efficient method for the detection of highly acidic multiply phosphorylated peptides and sulfopeptides in a background of tryptic peptides typically examined in most proteomic studies. Under typical positive ion mode analysis, sulfopeptides undergo proton mediated loss of the PTM, hampering identification and detection. Alkylamines were found to selectively adduct to sulfopeptides in positive ion mode nESI, allowing for discernment of isobaric phosphorylation and sulfation PTMs without the need for high resolution instrumentation. Alkylamine ion-pairing occurs at 98-99% efficiency regardless of solution pH or base concentration. Characteristic [SO3+alkylamine] neutral losses are observed upon slight collisional activation. This unique transition enables sulfopeptide identification and discovery with positive ion mode data-independent nLC tandem MS. Experiments to discover four sulfopeptide standards in a background of tryptic peptides resulted in 17 sulfopeptide identifications. To our knowledge, the work presented is the first protocol developed for positive ion mode sulfopeptide discovery without the need for tedious chemical modification of a sample proteome prior to analysis. Optimization of tryptic digestion and separation conditions were imperative for MS detection of covalently tethered intermediates in the polyketide synthase (PKS) bryostatin A, module 3 (BryAM3), which introduces a unique beta-branch and O-methylation in the biosynthesis of bryostatin-1, a potent protein kinase C inhibitor. BryAM3 was successfully (98%) phosphopantetheinylated to generate holo acyl carrier protein (ACP). Malonyl extender unit loading on holo ACP was also successfully achieved (68%) utilizing a non-native kirromycin C trans acyl transferase (KirCAT). Unexpectedly, KirCAT also catalyzed malonation of BryAM3 non-active site cysteine residues and direct ACP loading of thiophenol-activated substrates was observed. These experiments indicate that great care must be taken when performing in vitro studies with this and potentially other trans PKSs.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140863/1/mcclory_1.pd