The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with mnitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared to that obtained by CID for all charge states investigated. Multiply charged, N-terminally derivatized peptides were subjected to electron transfer reactions to produce singly charged, radical species. Upon subsequent "soft" CID, highly abundant z-type ions were formed nearly exclusively, which yielded simplified fragmentation patterns amenable to de novo sequencing methods. Furthermore, the simplified series of z ions were shown to retain labile phosphoric acid moieties. Infrared multiphoton dissociation (IRMPD) was implemented in a novel dual pressure linear ion trap for rapid "top-down" proteomics. Due to secondary dissociation, IRMPD yielded product ions in significantly lower charge states as compared to CID, thus facilitating more accurate mass identification and streamlining product ion assignment. This outcome was especially useful for database searching of larger proteins (~29 kDa) as IRMPD substantially improved protein identification and scoring confidence. Also, IRMPD showed an increased selectivity towards backbone cleavages N-terminal to proline and C-terminal to acidic residues (especially for the lowest precursor charge states). Ultraviolet photodissociation (UVPD) at 193 nm was implemented on a linear ion trap mass spectrometer for high-throughput proteomic workflows. Upon irradiation by a single 5 ns laser pulse, efficient photodissociation of tryptic peptides was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions. The factors that influence the UVPD mass spectra and subsequent in silico database searching via SEQUEST were evaluated. 193 nm ultraviolet photodissociation (UVPD) was employed to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. LC-MS/UVPD analysis using high pH mobile phases yielded efficient characterization of acidic peptides from mitogen-activated protein kinases.

PROVIDES STRATEGIES AND CONCEPTS FOR UNDERSTANDING CHEMICAL PROTEOMICS, AND ANALYZING PROTEIN FUNCTIONS, MODIFICATIONS, AND INTERACTIONS—EMPHASIZING MASS SPECTROMETRY THROUGHOUT Covering mass spectrometry for chemical proteomics, this book helps readers understand analytical strategies behind protein functions, their modifications and interactions, and applications in drug discovery. It provides a basic overview and presents concepts in chemical proteomics through three angles: Strategies, Technical Advances, and Applications. Chapters cover those many technical advances and applications in drug discovery, from target identification to validation and potential treatments. The first section of Mass Spectrometry-Based Chemical Proteomics starts by reviewing basic methods and recent advances in mass spectrometry for proteomics, including shotgun proteomics, quantitative proteomics, and data analyses. The next section covers a variety of techniques and strategies coupling chemical probes to MS-based proteomics to provide functional insights into the proteome. In the last section, it focuses on using chemical strategies to study protein post-translational modifications and high-order structures. Summarizes chemical proteomics, up-to-date concepts, analysis, and target validation Covers fundamentals and strategies, including the profiling of enzyme activities and protein-drug interactions Explains technical advances in the field and describes on shotgun proteomics, quantitative proteomics, and corresponding methods of software and database usage for proteomics Includes a wide variety of applications in drug discovery, from kinase inhibitors and intracellular drug targets to the chemoproteomics analysis of natural products Addresses an important tool in small molecule drug discovery, appealing to both academia and the pharmaceutical industry Mass Spectrometry-Based Chemical Proteomics is an excellent source of information for readers in both academia and industry in a variety of fields, including pharmaceutical sciences, drug discovery, molecular biology, bioinformatics, and analytical sciences.

The utility of 193 nm ultraviolet photodissociation (UVPD) and negative electron transfer dissociation (NETD) for the characterization of peptide anions was systematically evaluated. UVPD outperformed NETD in nearly all metrics; however, both methods provided complementary information to traditional collision induced dissociation (CID) of peptide cations in high throughput analyses. In order to enhance the performance of NETD, activated ion negative electron transfer dissociation (AI-NETD) methods were developed and characterized. The use of low-level infrared photoactivation or collisional activation during the NETD reaction period significantly improved peptide anion sequencing capabilities compared to NETD alone. Tyrosine deprotonation was shown to yield preferential electron detachment upon NETD or UVPD, resulting in N - C[alpha] bond cleavage N-terminal to the tyrosine residue. LC-MS/MS analysis of a tryptic digest of BSA demonstrated that these cleavages were regularly observed under high pH conditions. Transmission mode desorption electrospray ionization (TM-DESI) was coupled with 193 nm UVPD and CID for the rapid analysis and identification of protein digests. Comparative results are presented for TM-DESI-MS/CID and TM-DESI-MS/UVPD analyses of five proteolyzed model proteins. In some cases TM-DESI/UVPD outperformed TM-DESI-MS/CID due to the production of an extensive array of sequence ions and the ability to detect low m/z product ions. 193 nm UVPD was implemented in an Orbitrap mass spectrometer for characterization of intact proteins. Near-complete fragmentation of proteins up to 29 kDa was achieved. The high-energy activation afforded by UVPD exhibited far less precursor ion charge state dependence than conventional methods, and the viability of 193 nm UVPD for high throughput top-down proteomics analyses was demonstrated for the less 30 kDa protein from a fractionated yeast cell lysate. The use of helium instead of nitrogen as the C-trap and HCD cell bath gas and trapping ions in the HCD cell prior to high resolution mass analysis significantly reduced the signal decay rate for large protein ions. As a result, monoclonal IgG1 antibody was isotopically resolved and mass accurately determined. A new high mass record for which accurate mass and isotopic resolution has been achieved (148,706.3391 Da ± 3.1 ppm) was established.

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.

This dissertation details innovations in mass spectrometry-based instrumentation that expand our ability to survey biopolymers, including proteins, peptides, and oligonucleotides, with increased sensitivity, speed, and depth. Chapter 1 provides an overview of mass spectrometry instrumentation at the leading edge of proteomics and for nucleic acid analysis. In Chapter 2, the optical-fiber enabled coupling of a mass spectrometer to an infrared laser improves proteomic characterization. For Chapter 3, electron transfer dissociation-based proteomics is investigated and inefficiencies of related ion-ion reactions are characterized. Chapter 4 introduces a novel nucleic acid sequencing technology with activated ion-negative electron transfer dissociation to profile RNA molecules and their modifications. In Chapter 5, tandem mass spectrometry comprehensively characterizes heavily-modified RNA therapeutics. Chapter 6 focuses on a proteome-wide analysis of the bacterial post-translational modification O-mycoloylation. In Chapter 7, a novel tandem mass spectrometry strategy, termed activated ion-tandem mass tags, is revealed, boosting quantitative reporter ion generation. Chapter 8 expands upon AI-TMT for quantitative single-cell proteomics, realizing sensitivity gains which drive a greater dynamic range of quantification. In Chapter 9, a novel mass spectrometer enables the one hour human proteome and we explore one minute proteome analyses, pushing the limits of high-throughput proteome analysis. Chapter 10 discusses emergent proteomic techniques and suggests future directions of the field.

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.