Browsing by Subject "Mass spectrometry"

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Advanced development of ambient ionization mass spectrometry imaging methods and their utilization towards the understanding of metabolic diseases
(2020-07-06) Feider, Clara Leigh; Eberlin, Livia Schiavinato; Brodbelt, Jennifer S; Crooks, Richard; Milner, Thomas
Ambient ionization mass spectrometry (MS) technologies offer a unique opportunity to bring the specificity and sensitivity of MS into clinics, potentially allowing rapid analyses of biospecimens without need for sample preparation or highly trained personnel. As these technologies offer significant advantages over current technologies in terms of speed and suitability for a hospital environment, ambient ionization methods have been applied to a wide variety of clinical applications such as disease diagnosis, therapeutic drug monitoring, and biomarker discovery. Despite these successes, there remain challenges for these methods to overcome before they can be relied upon for routine analysis of patient samples. Prior to their use by physicians to inform decisions regarding patient care, ambient ionization MS technologies must be robust, versatile, and be shown to improve patient outcomes by providing information that is lacking in current standard of care procedures. This dissertation serves to outline recent advancements made towards the improvements of ambient ionization MS technologies as well as the application of these techniques towards disease indications that have yet to be explored. Chapter 2 provides data and discussion about how relative humidity can contribute to variability and poor data quality during desorption electrospray ionization (DESI) MS experiments, providing essential information about how atmospheric conditions can contribute to the robustness of the technique. Chapters 3 and 4 present methods for integration of new separation and fragmentation techniques with ambient ionization methods in order to increase the amount of information that can be gathered from a biological specimen. Finally, Chapter 5 presents results from the utilization of DESI-MS imaging towards understanding endometriosis, a chronic gynecological condition that has limited diagnosis and treatment options for patients. The entirety of this work aims to discuss how further improvements to ambient ionization MS technologies can make them useful for understanding a wider variety of disease processes and the progress that is being made to this end.
Advancement of photodissociation and electron-based tandem mass spectrometry methods for proteome analysis
(2011-08) Madsen, James Andrew; Brodbelt, Jennifer S.; Dalby, Kevin N.; Marcotte, Edward M.; Webb, Lauren J.; Willets, Katherine A.
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.
Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications
(2016-05) Robinson, Michelle Renee; Brodbelt, Jennifer S.; Crooks, Richard M; Dalby, Kevin N; Webb, Lauren J; Zhang, Yan J
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.
Advances and application of positive matrix factorization for source attribution of air pollution in megacities
(2021-02-22) Bhandari, Sahil; Hildebrandt Ruiz, Lea; Apte, Joshua S.; Sharma, Mukul M; Allen, David T
Air pollution is considered the greatest current environmental health threat to humanity, with an estimated mortality burden of 7 million per year. More than half the world’s population is exposed to increasing air pollution. Reduction of air pollution is essential to global health and can be expected to generate long-term societal benefits. Receptor models are efficient mathematical tools for identification of sources of air pollution. A popular receptor modeling technique is Positive Matrix Factorization (PMF). However, PMF is limited by the assumption of constant source profiles throughout the modeling period—while the contribution of each source is modeled to change over time, its profile (e.g., mass spectrum, when PMF is applied to mass spectrometer data) stays constant. PMF is frequently applied to data on air pollution from fine particulate matter (PM), particularly in megacities. Megacities are centers of economic activity, harbor very large populations, and have high PM levels, especially in the developing world, posing acute challenges to public health. One such city is Delhi, India. Delhi is the second most populated city in the world and routinely experiences some of the highest particulate matter concentrations of any megacity on the planet. However, the current understanding of the sources and dynamics of PM pollution in Delhi is limited. Measurements at the Delhi Aerosol Supersite (DAS) provide long-term chemical characterization of ambient submicron aerosol in Delhi, with near-continuous online measurements of aerosol composition. In this dissertation, I apply PMF on data collected in the DAS study to characterize sources and atmospheric dynamics of submicron aerosols in Delhi. In study 1 (chapter 2), I report on source apportionment based on unsupervised (unconstrained) positive matrix factorization (PMF), conducted on 15 months of highly time-resolved speciated submicron non-refractory PM₁ (NR-PM₁) between January 2017 and March 2018. This dataset was collected in the DAS study. I report on seasonal variability across four seasons of 2017 and interannual variability using data from the two winters and springs of 2017 and 2018. I also show that a modified tracer-based organic component analysis provides an opportunity for a real-time source apportionment approach for organics in Delhi. Phase equilibrium modeling of aerosols using the extended aerosol inorganics model (E-AIM) predicts equilibrium gas-phase concentrations and allows evaluation of the importance of the ventilation coefficient (VC) and temperature in controlling primary and secondary organic aerosol. I also find that primary aerosol dominates severe air pollution episodes, and secondary aerosol dominates seasonal averages. An edited version of this chapter has been published in Atmospheric Chemistry and Physics. In study 2 (chapter 3), we develop the approach of conducting supervised (constrained) PMF on long-term datasets separated into 4 hour periods with limited variability in emissions and meteorology and statistically demonstrate its viability. I apply this time-of-day PMF approach on two seasons of highly time-resolved NR-PM₁ organics. This approach improves upon the seasonal source apportionment previously employed in Delhi by capturing the diurnal variability in source mass spectral profiles and retaining low computational intensity. Use of the EPA PMF tool allows application of constraints and quantifies random errors and rotational ambiguity in PMF solutions. Results in this study demonstrate that time-of-day PMF approach gives a greater number of more appropriate PMF factors compared to the traditional seasonal PMF approach. The time-of-day PMF approach fits data better, improving fits at specific time points, and at key m/zs. Portions of this chapter will be submitted to Atmospheric Measurement Techniques. Previous receptor modeling studies have identified vehicular emissions and fossil fuel combustion as prevalent factors contributing to fine PM pollution in Delhi. However, cooking and biomass burning have not been consistently identified in ambient studies. Bottom-up (source-oriented) studies have recognized the high exposure to residential energy emissions from cooking and heating and associated biomass burning emissions. In study 3 (chapter 4), I address these limitations of receptor modeling studies by applying PMF on two seasons of highly time-resolved NR-PM₁ organics. I utilize the time-of-day PMF approach (chapter 3) to separate primary organics into component primary factors. Hydrocarbon-like organic aerosol, or HOA, the fuel combustion and traffic primary organic aerosol surrogate, occurs in every season, and shows strong diurnal patterns. Biomass burning organic aerosol, or BBOA, separates only in winter, and exhibits time series peaks associated with space heating and solid-fuel combustion. Cooking organic aerosol, or COA, separates only in monsoon and reports stable diurnal patterns, suggesting the presence of cooking sources all-day. Equilibrium modeling of organic aerosols using volatility basis sets (VBS) suggests that differences in ventilation coefficient and temperature can explain the differences in factor separation between winter and monsoon. Overall, I show that traffic, and cooking and biomass burning contribute almost equally to the primary organic aerosol burden in Delhi, in broad agreement with several bottom-up studies. Portions of this chapter will be submitted to Atmospheric Chemistry and Physics
Algorithms for biomarker identification utilizing MALDI TOF mass spectrometry
(2006) Shin, Hyunjin; Valvano, Jonathan W., 1953-; Markey, Mia Kathleen
Currently, the best way to reduce the mortality of cancer is to detect and treat it in the earliest stages. Technological advances in genomics and proteomics have opened a new realm of methods for early detection that show potential to overcome the drawbacks of current strategies. In particular, pattern analysis of mass spectra of blood samples has attracted attention as an approach to identification of potential biomarkers for early detection of cancer. Mass spectrometry provides rapid and precise measurements of the sizes and relative abundances of the proteins present in a complex biological/chemical mixture. However, this high-throughput nature of mass spectrometry has also raised a need for the development of efficient and effective bioinformatics tools for finding biologically meaningful information. Many scholars are interested in preprocessing of raw mass spectra and in extracting and selecting features from preprocessed mass spectra. These are key issues for accurate biomarker identification. Thus, in order to improve the process of biomarker identification using mass spectrometry, I have postulated a noise model for MALDI TOF mass spectrometry from the perspective of stochastic signal processing, and have attempted to measure the spectral characteristics of components in the noise model. Noise in mass spectrometry can interfere with identification of the biochemical substances in a sample. I assumed that the noise in MALDI TOF mass spectrometry is composed of three components: noise from instrumentation, noise from random ion motions, and chemical noise. In this dissertation, I have separated and analyzed noise from instrumentation and chemical noise using parametric power spectral density estimation and wavelet-based analysis, respectively. In addition to these noise analysis studies, I also have designed an algorithm that can select independent and discriminant features from mass spectra of complex protein samples by reducing redundant and irrelevant information.
Anthropogenic influence on the fate of secondary organic aerosol : chlorine chemistry and alkane oxidation
(2022-10-06) Wang, Dongyu; Hildebrandt Ruiz, Lea; Allen, David T; Donahue, Neil M; Rochelle, Gary T; Xu, Ying
Oxidation of volatile organic compounds (VOC) in the atmosphere leads to the formation of secondary organic aerosol (SOA), which can have extensive impacts on air quality, health, and climate. Existing air quality models used to describe the fate of ambient organic aerosol tend to underpredict the aerosol oxidation state. In addition, modeled concentrations of nitrogen oxides (NO [subscript x]) and other reactive nitrogen compounds (NO [subscript y]), including alkyl nitrates, often deviate from field observations. Certain SOA formation pathways, SOA ageing mechanisms, and alkyl nitrate decay mechanisms may be missing. Recent field studies show that NO [subscript x]-mediated heterogeneous production of nitryl chloride, ClNO₂, could provide a ubiquitous source for chlorine atoms. Little is known about the role of chlorine atoms in SOA formation and ageing, or their interaction with other anthropogenic emissions found in polluted environments, where alkane oxidation chemistry is important. Environmental chamber experiments are carried out to address knowledge gaps in atmospheric chlorine and alkane oxidation chemistry. Results show that chlorine-initiated oxidation of isoprene leads to SOA formation, organic chloride formation, and possibly secondary HO [subscript x] chemistry. Alkane-derived alkyl nitrate compounds are found not to hydrolyze appreciably in humid environments or in the presence of acidic aerosol. Uptake of inorganic nitrate and inorganic chloride are observed in the presence of deliquescent particles. Chlorine-initiated oxidation of linear alkanes is shown to result in prompt SOA formation and delayed organic chloride formation, which is enabled by the addition of chlorine radical to dihydrofuran, a heterogeneously produced multi-generational oxidation product. Improvements are made for the detection of organic chloride using aerosol mass spectrometry, and for aerosol volatility measurements using temperature programmed thermal desorption techniques. A two-dimensional thermogram framework is developed to visualize aerosol composition, aerosol volatility, and thermal fragmentation simultaneously.
Applications of desorption electrospray ionization mass spectrometry imaging for disease characterization from tissue sections and minimally invasive biopsies
(2022-05) Bensussan, Alena; Brodbelt, Jennifer S.; Eberlin, Livia Schiavinato; Webb, Lauren; Cressman, Erik
Due to improved therapeutic regimens for cancer, disease grade, stage, and subtype, have become pertinent stratifications for prescribing an optimized treatment plan for every patient. Histopathologists are now being asked to perform increasingly complex disease distinctions from small biopsy samples that may or may not provide the necessary information to make such distinctions. Since inception, mass spectrometry (MS) have been proven to be a powerful analytical tool for disease diagnosis and several MS techniques have been successfully integrated into routine clinical workflows. Over the past two decades, ambient ionization MS, particularly desorption electrospray ionization mass spectrometry imaging (DESI-MSI), has been studied for disease differentiation in a similar manner. Intrinsic advantages of DESI-MSI, such as minimal sample preparation, nondestructive solvent system, and spatial separation of relevant tissue, supports the successful integration of this technology into a pathological workflow. Despite these advantages, research is ongoing to determine the efficacy of DESI-MSI as a tool for intricate disease stratification and biopsy analysis. This dissertation presents the applications of DESI-MSI towards staging advanced disease, classifying preneoplastic lesions, and subtyping cancerous tissue from tissue sections and minimally invasive biopsy material. Chapter 2 discusses the application of DESI-MSI and statistical analyses to understand metabolic dysregulation in primary and metastatic melanoma as well as discusses the performance of statistical classifiers on metastatic melanoma in lymph node tissue. Chapter 3 presents the application of DESI-MSI and statistical classifiers towards differentiation of low grade and high grade preneoplastic pancreatic cysts. Finally, Chapter 4 describes the application of this technique towards the stratification of lung cancer subtypes from tissue sections and biopsy material. In entirety, this work aims to demonstrate the capabilities of the DESI-MSI workflow towards increasingly complex biospecimens and diagnostic challenges commonly confronted in routine clinical environments.
Biopolymer analysis by electrospray ionization and tandem mass spectrometry
(2004) Keller, Karin Mia; Brodbelt, Jennifer S.
Electrospray ionization was used in conjunction with Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry and quadrupole ion trap (QIT) mass spectrometry to study protein and oligonucleotide ions in vacuo. The results help to identify effective strategies for mass spectral analysis of these macromolecules and provide new insight on their gas-phase behavior. Tandem mass spectrometry experiments were conducted to evaluate different ion activation methods for biopolymer sequencing. Multipole storage-assisted dissociation (MSAD) and sustained off-resonance irradiation collision-activated dissociation (SORI- CAD) were compared for protein analysis in FTICR instrumentation, and infrared multiphoton dissociation (IRMPD) and collisonal activated dissociation (CAD) were compared for oligonucleotide analysis in QIT instrumentation. In both studies, the differences in the observed fragmentation patterns were noted and the underlying reasons for these differences were identified. The relative utility of MSAD vs. SORI-CAD and IRMPD vs. CAD were assessed in terms of their ability to produce diagnostic information that could be used to identify the protein or oligonucleotide under study. Tandem mass spectrometry was also employed to study the dissociation patterns of both DNA/metal and DNA/drug complexes. The preferred fragmentation pathways exhibited by these species were observed to vary with the initial charge state of the precursor. The effect of the oligonucleotide sequence, the identity of the metal ion, and the identity of the drug on these pathways was established and (where possible) interpreted in terms of the specific non-covalent bonding patterns present in the parent complexes. Finally, electrospray ionization was evaluated as a tool for screening molecular recognition in nucleic acid aptamer/small molecule interactions. Gas-phase data for binding stoichiometry and relative binding affinity were compared with the known solution behavior for a series of well-characterized case studies. Any observed discrepancies were rationalized in terms of ligand structure and/or the nature of the intermolecular ligand/aptamer interactions.
Characterization and isomer differentiation of glycosides and oligosaccharides using chemical derivatization with quadrupole ion trap mass spectrometry
(2007-12) Pikulski, Michael, 1969-; Brodbelt, Jennifer S.
Several innovative tandem mass spectrometric strategies have been developed for the structural determination and isomer differentiation of glycosides and oligosaccharides. Specifically, collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) are used in conjunction with derivatization methods designed to exploit variations in binding energies or attach chromophores. These include metal complexation incorporating modified neutral auxiliary ligands and covalent derivatization involving site-specific reactions. The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and CAD of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]⁺ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. We compare and explore the use of alternative pyridyl ligands, with electron-releasing substituents including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin and luteolin. A tunable ESI-MS/MS strategy for differentiation of flavone and flavanone diglycoside isomers based on metal complexation with auxiliary ligands that have electron-withdrawing substituents is reported. A series of auxiliary ligands with electronwithdrawing substituents was synthesized in order to tailor the relative metal binding affinities of the ligands and thus directly influence the stabilities, and consequently the dissociation pathways, of the complexes. Upon collisionally activated dissociation, the complexes yield fragmentation patterns in which the abundances of key diagnostic ions are enhanced, thus facilitating isomer differentiation. A strategy for increasing the efficiency of IRMPD in a quadrupole ion trap (QIT) based on another metal complexation strategy is described. Two IR-active ligands (IRALs) that have an IR-active phosphonate functional groups were synthesized. The IR-active groups were therefore incorporated into the analyte complexes via metal complexation. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energyvariable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands. A simplified method for determining the sequence and branching of oligosaccharides using IRMPD in a QIT is described. An IR-active boronic acid (IRABA) reagent was synthesized and subsequently used to derivatize the oligosaccharides prior to IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. The approach is demonstrated for two series of oligosaccharides, the lacto-Nfucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).
Characterization of peptides and proteins by ultraviolet photodissociation mass spectrometry for biotechnology applications
(2023-12) Watts, Eleanor (Ph. D. in chemistry); Brodbelt, Jennifer S.; Georgiou, George; Brenna, Thomas J; Ellington, Andrew D
Given the rapid and ubiquitous growth of biotechnology as a field, expanding the capabilities for the characterization of peptides and proteins remains paramount. Extending the breadth of applications for advanced tandem mass spectrometry methods has been critical to improving the understanding of biomolecules. The research presented in this dissertation focuses on the characterization of peptides and proteins with ultraviolet photodissociation-mass spectrometry (UVPD-MS). In addition to enhancing the body of knowledge on the applications of UVPD, this results in improved tools to characterize biotherapeutics and other biomolecules relevant to the biotechnology industry, including antibody drug conjugates (ADCs), synthetic selenoproteins, and human leukocyte antigen (HLA) immunopeptides. Through this work, improved localization of drug conjugation sites is presented for both cysteine- and lysine-linked ADCs, complete characterization of sequence and diselenide bond locations is reported for synthetic selenoproteins, and comprehensive sequence information, including leucine and isoleucine differentiation, is established for HLA immunopeptides. Various sample preparation techniques are explored to suit each application, including bottom-up and middle-down proteolysis as well as top-down analysis. High resolution mass spectrometry instrumentation and modern tandem mass spectrometry platforms are integral to the research presented in this dissertation. In addition to UVPD, a variety of tandem mass spectrometry techniques are explored including collisional and electron transfer dissociation. External collaborations have contributed greatly to the impact of this work, ensuring the development of industry-relevant applications. Access to application-specific software suits has also facilitated and enhanced the analysis and presentation of high-quality data. Overall, the work represents a step forward in both the understanding of key biomolecules as well as the applicability of UVPD to key areas in biotechnology research.
Characterization of protein post-translational modifications using 193 nm ultraviolet photodissociation mass spectrometry
(2023-12) Bashyal, Aarti; Brodbelt, Jennifer S.; Webb, Lauren J.; Paull, Tanya; Eberlin, Livia S.
Protein characterization remains the highest-impact application and one of the critical challenges for mass spectrometry. The dissertation begins with a fundamental investigation of how charging methods affect the dissociation patterns of intact proteins in low charge states when using UVPD compared to higher energy collisional dissociation (HCD). UVPD outperforms HCD in efficiently fragmenting proteins in low charge states irrespective of the charging method. Next, the utility of 193 nm ultraviolet photodissociation (UVPD) to localize protein post-translational modifications (PTMs) was explored. The isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) is a critical PTM that can significantly influence the quality and efficacy of protein-based pharmaceuticals. The ability of 193 nm UVPD to distinguish Asp and isoAsp in the protonated and deprotonated peptides was investigated. The differences in the relative abundances of fragment ions uniquely generated by UVPD were leveraged to differentiate isomeric peptides containing Asp or isoAsp and to identify an isomerized peptide from a tryptic digest of a monoclonal antibody. Ubiquitination, a PTM in which ubiquitin molecules are added to substrate proteins via iso-peptide linkage, was also studied. Multiple ubiquitin chains can be added to the N-terminus or one of the seven lysines of the ubiquitin in linear or branched chains, forming complex polyubiquitin chains with distinct biological functions. UVPD, followed by gas-phase proton transfer charge reduction (PTCR), was used to localize the linkage sites in six linearly linked tetraubiquitin standards. UVPD generates a diverse array of sequence ions, resulting in congested spectra due to the overlapping isotopic distribution of highly charged fragment ions for proteins >30 kDa. PTCR was employed to decongest the resultant spectra, facilitating the assignment of previously obscured product ions. A comprehensive literature review was also undertaken to highlight the current status and future directions in analyzing low-abundance PTMs. This review emphasizes the need for advancements in mass spectrometry-based techniques to characterize PTMs like ADP-ribosylation, tyrosine sulfation, and nitration. In the final chapter, UVPD, in conjunction with native mass spectrometry, was utilized to probe the residues modified by two novel covalent inhibitors of New Delhi metallo-β-lactamase 1 (NDM1), a class B1 broad-spectrum MBL that can bind and hydrolyze almost all β-lactam antibiotics.
Characterization of proteins and peptides via enhanced 266 nm ultraviolet photodissociation mass spectrometry utilizing a selenium based chromophore
(2016-05) Parker, William Ryan; Brodbelt, Jennifer S.; Eberlin, Livia S.
Mass spectrometry and chemical derivatization have been used as tools for the identification of proteins in both top-down and bottom-up studies. Cysteine is the rarest and most nucleophilic amino acid thus making it a popular target for chemical tagging strategies. Ultraviolet photodissociation (UVPD) is a versatile activation technique for fragmentation of peptides and proteins. For successful photodissociation, ions of interest must contain a suitable chromophore that matches the wavelength of irradiation. N-(Phenylseleno)phthalimide (NPSP) is a fast reacting reagent which attaches a selenium based chromophore that absorbs at 266 nm light to free thiols. In the studies presented in this thesis, NPSP was used to derivatize free cysteine residues in both intact proteins and tryptic peptides. Activation with 266 nm photons causes a dominant neutral loss of the benzeneselenol groups on the tagged protein or peptide ions. This diagnostic neutral loss allows the determination of the number of free versus bound cysteine residues in intact proteins. Additionally, tagging peptides with benzeneselenol provides a means to target only the cysteine-containing peptides in bottom-up proteomics experiments. Both of these methods provide a significantly reduced search space for identification of cysteine-containing proteins. Counting the number of cysteine residues also provides an effective way to restrict the number of protein candidates for database searches. Moreover, cysteine peptides are inherently more unique than other peptides created upon enzymatic digestion of proteins due to the low frequency of cysteine in the proteome, thus allowing these peptides to be used as surrogates for protein identification.
Computational methods in protein mass spectrometry, DNA microarray technology and protein folding
(2005) Nakorchevskiy, Aleksey Alfred; Marcotte, Edward M.
Bottom-up protein tandem mass spectrometry is one of the most widely used high-throughput methods in proteomics and generally relies on the identification of proteins from the masses and fragmentation patterns of their proteolytic peptides. Using this approach, peptides are identified through spectral alignment of the experimental peptide fragmentation spectra to in silico-generated peptide fragmentation spectra. We introduce a probabilistic framework where each protein’s identification is dependent on several independent types of data such as precursor ion charge, precursor ion mass, MS2 alignment, and chromatographic alignment. We construct a parallel architecture platform ProteinFinder where we investigate various scoring schemes for these types of data and establish their relative contributions towards the overall protein identification. In addition, we propose an approach that allows for protein identification from highly complex biological samples from the full scan data alone, or that may be used in conjunction with MS2 data to provide additional interpretative power. The Peptide Signature Method (PMS) analyzes correlations of peptide abundances across multiple experiments, exploiting the fact that peptides derived from the same protein should be present stoichiometrically, and therefore their concentrations will correlate as the protein’s concentration changes. By comparing mass spectral peaks from several independent mass spectrometry experiments, peptides are clustered by the pattern of their abundances (“peptide signatures”) throughout the experiments. Proteins are then identified via peptide mass fingerprinting of the peptide co-expression clusters. We also apply a method called Expression Deconvolution to deconvolute the DNA microarray expression data and study relative contributions of different multiple myeloma pure expression programs towards the samples from mixed population of newly diagnosed multiple myeloma patients and multiple gammopathy of undetermined significance. Finally, to gain a better understanding of the determinants of protein folding rates, we apply the method of relative contact order to predict folding rates of proteins with known tertiary structure that are classified according to CATH hierarchy. We survey the PDB database and establish the hierarchy levels that determine the protein folding properties. We also estimate the theoretical range of the folding rates for the single domain proteins and probe individual protein structures for fast and slow folding regions.
De novo sequencing and peptide characterization via ultraviolet photodissociation mass spectrometry
(2015-05) Robotham, Scott Allen; Brodbelt, Jennifer S.; Crooks, Richard M; Ellington, Andrew; Shear, Jason B; Webb, Lauren
Although in silico database search methods remain more popular for shotgun proteomics methods, de novo sequencing offers the ability to identify proteins lacking sequenced genomes and ones with subtle splice variants or truncations. Ultraviolet photodissociation (UVPD) of peptides derivatized by selective attachment of a chromophore at the N-terminus generated characteristic series of y ions. The UVPD spectra of the chromophore-labelled peptides were simplified and thus amenable to de novo sequencing. E.coli lysates were modified by the use of carbamylation and the attachment of a UV chromophore to the N-terminus of digested peptides. UVPD of the resulting peptides generated clean sets of y ions. A novel de novo algorithm, UVnovo, afforded high confidence identification of thousands of peptides from an E. coli lysate and allowed UVPD/CID paired spectra to be searched. E.coli lysate peptides were analyzed in alternating scans by UVPD and CID on the same precursors to generate paired UVPD/CID spectra. UVnovo enabled sequence tag de novo sequencing of peptides in order to find matching sequences from a database. Ultimately the UVnovo functioned as a standalone de novo sequencing program or a hybrid de novo/database search program. In an effort to better characterize the fragmentation pathways promoted by ultraviolet photoexcitation in comparison to CID, six adrenocorticotropic hormone (ACTH) peptides in a range of charge states were subjected to 266 nm ultraviolet photodissociation (UVPD), 193 nm UVPD, and CID. While both UVPD and CID led to preferential cleavage of the Y-S bond for all ACTH peptides (except ACTH (1-39)), UVPD was far less dependent on charge state and location of basic sites for the production of C-terminal and N-terminal ions.
Development and application of methods towards the structural characterization of gas-phase biomolecular assemblies
(2022-05-05) Sipe, Sarah Natalie; Brodbelt, Jennifer S.; Eberlin, Livia S.; Webb, Lauren J.; Whitman, Christian P.
The utility of ultraviolet photodissociation mass spectrometry (UVPD-MS) in native MS approaches, including ion mobility spectrometry (IMS), for protein complexes is described in this dissertation. A modular drift tube demonstrated suitability for measuring collision cross sections (CCSs) of native-like ions on an Orbitrap mass spectrometer with high resolution using acquisition times as short as one minute. This IMS method is used throughout this dissertation for measurement of native-like and disordered structures. A fundamental study for determining the charge-dependent behavior of UVPD for protein complexes was evaluated using the homomeric Cu/Zn superoxide dismutase dimer, streptavidin tetramer, transthyretin tetramer, and C reactive protein pentamer as well as the heteromeric hemoglobin tetramer. A wide range of charge states were irradiated with 193 nm photons resulting in asymmetric charge partitioning of subcomplexes at lower energies (0.5 to 1.5 mJ/pulse) and symmetric dissociation at higher energies (1.5 to 3.0 mJ/pulse). The ability to access both of these competing dissociation pathways is unique to UVPD and contributes to the vast array of sequence ions and enhanced sequence coverage for protein complexes not obtained by any other activation method. With its ability to generate useful sequence information, UVPD was employed to study an intrinsically disordered protein, a set of asymmetric and symmetric trimers, and three membrane protein complexes. The vast population of structures adopted by the intrinsically disordered protein, high mobility group protein AT-hook 2 (HMGA2), was characterized using UVPD and the probable binding location of two DNA hairpins was determined. Trimers in the tautomerase superfamily that have nearly identical secondary structures differ in their quaternary arrangements to form asymmetric and symmetric homooligomers. In combination with collision-induced unfolding, UVPD proved capable of differentiating the two structures owing to the preservation of noncovalent interactions in the gas phase. Aquaporin z (AqpZ), mechanosensitive channel of large conductance (MscL), and the E. coli ammonia channel (AmtB) comprise the membrane protein complexes studied herein. UVPD of these complexes resulted in unprecedented levels of characterization with backbone cleavages demonstrating no significant influence from the hydrophobicity of the residues or the mobile proton-directed cleavages, which contrasts reports using electron- and collision-based dissociation methods, respectively. UVPD has also proven effective for localizing phosphorylated residues along the C-terminal domain (CTD) of RNA polymerase II, shedding light on the CTD code that mediates transcription regulation.
Development of chromogenic cross-linkers and selective gas-phase dissociation methods to assess protein macromolecular structures by mass spectrometry
(2009-12) Gardner, Myles Winston; Brodbelt, Jennifer S.; Marcotte, Edward M.; Shear, Jason B.; Stevens, Scott W.; Stevenson, Keith J.
Selective gas-phase dissociation strategies have been developed for the characterization of cross-linked peptides and proteins in quadrupole ion trap mass spectrometers. An infrared chromogenic cross-linker (IRCX) containing a phosphotriester afforded rapid differentiation of cross-linked peptides from unmodified ones in proteolytic digests of cross-linked proteins by selective infrared multiphoton dissociation (IRMPD). Only the cross-linked peptides containing the chromogenic phosphate underwent IRMPD and unmodified peptides were not affected by IR irradiation. IRMPD of IRCX-cross-linked peptides yielded uncross-linked y-ion sequence tags of the constituent peptides due to secondary dissociation of all primary product ions which contained the chromophore, thus allowing successful de novo sequencing of the cross-linked peptides. Peptides cross-linked via a two-step conjugation strategy through the formation of a bis-arylhydrazone (BAH) bond were selectively dissociated by ultraviolet radiation at 355 nm. The BAH-cross-linked peptides could be distinguished from not only unmodified peptides but also dead-end modified peptides based on the selectivity of ultraviolet photodissociation. In a complementary approach, electron transfer dissociation of BAH-cross-linked peptides resulted in preferential cleavage of the hydrazone bond which produced two modified peptides. These modified peptides were subsequently interrogated by CID which allowed for the original site of cross-linking to be pinpointed. IRMPD was implemented in a dual pressure linear ion trap to demonstrate successful photodissociation of peptides having modest absorptivities. Peptides were observed to efficiently dissociation by IR irradiation exclusively in the low pressure cell whereas no dissociation was observed in the high pressure cell due to extensive collisional cooling. IRMPD provided greater sequence coverage of the peptides than CID and yielded product ion mass spectra which were predominantly composed of singly charged product ions which simplified spectral interpretation. IRMPD was further applied for the sequencing of small-interfering RNA. Complete sequence coverage was obtained and the results were compared to CID.
Development of mass spectrometric methods for proteomics analysis utilizing gas-phase chemistry and ultraviolet photodissociation
(2016-10-20) Holden, Dustin Donald; Brodbelt, Jennifer S.; Mullins, Charles B.; Roberts, Sean T.; Eberlin, Livia S.; Baiz, Carlos R.
The utility of ultraviolet photodissociation (UVPD) in comparison to higher-energy collisional dissociation (HCD) to provide sequence coverage was assessed for various protein cation charge states and sizes. UVPD provided consistently higher sequence coverages through more uniform fragment ion distribution along the protein sequence. HCD provided lower sequence coverage values as well as more preference towards cleavage at the most labile bonds. Assessment of coverage dependence at lower charge states was also performed through proton transfer reactions (PTR) with ion parking. Overall, HCD provided preferential cleavage C-terminal to amino acids with acidic sidechains and N-terminal to proline, while UVPD provided more evenly distributed cleavage sites with enhancement near proline and phenylalanine. Using UVPD as a structural analysis tool, PTR was assessed for perturbations to native-like structure of various protein complexes. Through comparison of UVPD fragment intensities, spectra of protein complexes generated through PTR showed little difference to spectra obtained from native-like protein spectra. Following this, PTR-UVPD was applied to elucidate fragment origins of an ambiguous homodimeric protein complex, otherwise displaying complex a complex mass spectrum of overlapping species. A novel approach to performing UVPD using light emitting diodes (LEDs) was explored involving the engineering of a new planar ion trap. Commercially available ultraviolet LEDs emitting photons with wavelengths ranging from 255 to 275 nm were obtained and interfaced with the new ion trap. Sequestration of sample ions in a small spot allowed optimization of overlap with LED photons and resulting fragmentation efficiencies were assessed. Once optimized, LED-UVPD was successfully performed for electron photodetachment (EPD) of single stand DNA and tyrosine sidechain cleavage of a peptide. Custom instrument function was enabled to automatically resonantly eject un-dissociated precursor ions following UVPD and was applied during liquid chromatography (LC) bottom-up proteomics experiments. Through ejection of uninformative, un-dissociated precursor ions, detrimental mass shifting effects caused by increasing the number of charges during spectrum acquisition were relieved. It was observed that performing PE-UVPD resulted in higher protein identification confidence values than for UVPD alone for an E. coli whole cell lysate digest.
Development of mass spectrometry and ion mobility methods and instrumentation for the characterization of proteins from primary sequence to higher order structure
(2022-11-09) Sanders, James Daniel; Brodbelt, Jennifer S.; Eberlin, Livia S.; Marcotte, Edward M.; Baiz, Carlos R.
The development of methodology and instrumentation associated with mass spectrometry (MS) and ion mobility (IM) spectrometry for the characterization of proteins and other complex biomolecules was described and evaluated. 193 nm ultraviolet photodissociation (UVPD), when applied in both middle-down and top-down proteomic workflows, produced higher sequence coverage when compared to collisional activation methods. Using carbamylation of lysine to restrict tryptic cleavage to less common arginine residues, larger peptides were produced that benefited from the enhanced sequence coverage of UVPD to enable better characterization of protein sequence regions typically missed in traditional bottom-up workflows. When UVPD was combined with gas-phase charge reducing proton transfer reactions selectively applied to reduce spectral complexity, these gains were even more significant and enabled in-depth characterization of proteins as large as 56 kDa. MS and IM technologies were further applied to the characterization of higher order molecular structure. A novel method was developed to measure collision cross sections (CCS) of intact protein ions using the decay rate of the time domain transient signal produced by an Orbitrap mass analyzer. This method produced measurements with < 10% deviation from those generated by ion mobility without the need for specialized equipment or instrument modification. Methods to couple IM separations to Orbitrap mass spectrometry were developed and evaluated. The application of absorption mode processing to Fourier transform IM multiplexing techniques resulted in significant improvements in both resolution and signal to noise ratio compared to traditional magnitude mode processing. Performing UVPD on glycerophospholipids following IM separation enabled the accurate measurement of CCS from mixtures of double-bond isomers even when they cannot be fully resolved by standard IM instrumentation. Finally, a reduced pressure IM drift tube was designed and built to interface with a UVPD equipped Orbitrap mass spectrometer and used to characterize heterogeneous higher-order gas-phase protein structures using post IM UVPD.
Development of photodissociation methods for biomolecule analysis in a quadupole ion trap mass spectrometer
(2008-05) Wilson, Jeffrey John, 1979-; Brodbelt, Jennifer S.; Peppas, Nicholas A., 1948-
Photodissociation methods have been implemented and compared to collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer for the structural analysis of peptides, proteins, oligosaccharides, DNA and DNA/drug complexes. Infrared multiphoton dissociation (IRMPD) was applied to N-terminally sulfonated peptides which offers efficient photo-fragmentation and detection of important low m/z fragments in comparison to CID. Upon IRMPD of these modified peptides a simplified MS/MS spectrum comprised of only characteristic y ions allows for better identification through de novo software analysis. Oligonucleotides can undergo highly efficient IRMPD due to the phosphate moiety located on along their backbone structure which yields excellent photon absorption at [lambda] = 10.6 [mu]m. IRMPD fragmentation pathways of DNA and DNA/drug complexes were shown to be comparable to CID, yielding cleavage at the [w / (a - B )] bond, except IRMPD allows for significantly improved MS/MS sensitivity through the secondary dissociation of uninformative duplex base losses which can further dissociate into useful fragment ions for sequencing. Ultraviolet photodissociation (UVPD) has been applied to chromophorederivatized peptides and oligosaccharides which retains the advantages associated with IRMPD, but also has additional benefits due to the greater energy per photon at 355 nm (3.5 eV / photon) in comparison to 10.6 [mu]m (0.12 eV / photon). Primarily, UVPD provides highly efficient secondary dissociation of chromophore-containing fragments allowing for simplified MS/MS spectra of chromophore-derivatized peptides. This concept was also implemented for the characterization of branched fluorescently-labeled oligosaccharides which produces different fragment ions complementary to CID experiments. Secondly, UVPD provides an ion activation method which is independent of the bath gas helium pressure in the ion trap in contrast to CID or IRMPD permits for optimal trap performance without compromise. Coordination of a chromogenic 18-crown-6 molecule to the lysine side chain of a peptide facilitates UVPD at both 266 nm and 355 nm. Energy absorbed by the crown ether is transferred intermolecularly to the peptide via the strong hydrogen bonds which hold the complex together, resulting in activation and fragmentation of the peptide. CID or IRMPD of these crown ether/peptide complexes results only in their disassembly without peptide fragmentation.
Development of tandem mass spectrometric methods for proteome analysis utilizing photodissociation and ion/ion reactions
(2013-08) Shaw, Jared Bryan; Brodbelt, Jennifer S.
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.