Post-translational modifications (PTMs) are important for regulating protein structure and function. Despite significant progress for PTM analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS), opportunities for new method development remain. The research presented in this dissertation promotes 193 nm ultraviolet photodissociation (UVPD) as an alternative activation technique for PTM analysis with specific utility for phosphorylated and sulfated peptides. A novel de novo sequencing method with applications for unbiased PTM discovery was developed utilizing Lys-N proteolysis, N-terminal imidazolinylation, and UVPD to direct fragmentation for the formation of N-terminal ions. The N-terminal a, b, and c ions generated by UVPD were differentiated from one another by characteristic mass shifts. Sets of triplet peaks were used to distinguish N-terminal ions from confounding C-terminal ions and improve the accuracy of de novo sequencing. UVPD was evaluated for the analysis of phosphopeptide cations and anions. Negative mode analysis was advantageous for the detection of casein peptides in high phosphorylation states, while positive mode proved more robust for global phosphoproteomic analysis of HeLa and HCC70 cell lysates. Compared to collisional activation, the depth of coverage was lower using UVPD yet more extensive fragmentation and improved phosphate retention on products ions was achieved. Phosphorylation mapping by LC-UVPD-MS was carried out in the C-terminal domain (CTD) of RNA polymerase II as a function of kinase treatment, ERK2 or TFIIH, and organism, yeast or fruit fly. Single phosphorylations on Ser2 or Ser5 in the consensus heptad, YSPTSPS, were observed across all experimental conditions. Analysis of the non-consensus fruit fly CTD revealed the significance of Tyr1 and Pro residues in the +1 position relative to Ser for phosphorylation to occur. For sulfated peptides, negative mode UVPD yielded a and x ions that largely retained the labile sulfate modification, facilitating peptide sequencing and PTM localization. With appropriate MS/MS tools established, the next step towards global sulfoproteomics was the development of enrichment methods. Weak anion exchange (WAX) was applied for this purpose. Following carbamylation to neutralize primary amines which otherwise repel the anion exchanger; improved WAX retention was observed for sulfopeptides relative to a complex mixture of unmodified bovine serum albumin peptides.

Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.

Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.

This is an in-depth guide to the theory and practice of analyzing raw mass spectrometry (MS) data in proteomics. The volume outlines available bioinformatics programs, algorithms, and databases available for MS data analysis. General guidelines for data analysis using search engines such as Mascot, Xtandem, and VEMS are provided, with specific attention to identifying poor quality data and optimizing search parameters.

When the last edition of this book was published in 2000, the field of proteomics was in its infancy. At that time, multidimensional liquid chromatographic separations were being introduced as an alternative to traditional gel-based techniques for separating complex protein and peptide mixtures prior to mass spectrometric detection. Today, this approach – referred to as shotgun proteomics – is considered routine for lar- scale global analyses of protein mixtures. Now in its adolescence, proteomics is fundamentally transforming biological and medical research. Much of this transformation can be attributed to technological advancements, particularly in mass spectrometry. Much wider accessibility of hi- resolution and mass measurement accuracy instrumentation in recent years has ini- ated a new revolution in the field by providing more reliable data and shifting the focus from cataloging proteins to precisely quantifying changes in protein abundance over time and in response to stimuli. Advanced mass spectrometers and novel ion d- sociation schemes such as electron transfer/capture dissociation make it possible to venture boldly into the maze of protein posttranslational modifications, which are an integral component of understanding functional proteomics in the spatial and t- poral domains. Another area that has benefited from these advancements is top-down proteomics, an emerging method essential for characterizing various protein variants that has potentially high impact in biomedical research.