A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies

A key step in therapeutic and endogenous humoral antibody characterization is identifying the amino acid sequence. So far, this task has been mainly tackled through sequencing of B-cell receptor (BCR) repertoires at the nucleotide level. Mass spectrometry (MS) has emerged as an alternative tool for obtaining sequence information directly at the - most relevant - protein level. Although several MS methods are now well established, analysis of recombinant and endogenous antibodies comes with a specific set of challenges, requiring approaches beyond the conventional proteomics workflows. Here, we review the challenges in MS-based sequencing of both recombinant as well as endogenous humoral antibodies and outline state-of-the-art methods attempting to overcome these obstacles. We highlight recent examples and discuss remaining challenges. We foresee a great future for these approaches making de novo antibody sequencing and discovery by MS-based techniques feasible, even for complex clinical samples from endogenous sources such as serum and other liquid biopsies

Characterization of cellular stress systems using biological mass spectrometry

In recent years mass spectrometry has become an invaluable tool to address an array of biological questions. The versatility of this experimental approach does not only allow assignment of protein identity and identification of sequence specific modifications, but with the help of particular derivatization techniques facilitates the determination of peptide quantity. Each of these approaches were applied to the following biological projects: The 21 kDa heat stable protein purified from the encysted embryo of Artemia franciscana was characterized by time-of-flight mass spectrometry. De novo sequencing of peptides identified this protein as a group 1 Late embryogenesis abundant (LEA) protein. The amino acid sequence assignment to these peptides allowed amplification of the entire gene sequence from an embryonic cDNA library. This was deposited into the NCBI database (EF656614). The expression of group 1 LEA protein is consistent with and supports a role in desiccation tolerance. In addition, this is a first report describing identification of a group 1 LEA protein in an animal species. A MS-based quantitative analysis was performed in order to analyze relative changes in the dynamic thiol and disulfide states of the redox sensitive protein disulfide isomerase, PDI. PDI cysteine residues were derivatized with an isotope-coded affinity tag (ICAT), thus allowing a direct comparison between the reduced and auto-oxidized forms. Quantitation was based on relative ratios between light and heavy isotopically labeled cysteine containing peptides. The application of the ICAT-labeling approach to PDI related studies, allowed direct assignment of individual cysteine residues and their oxidation status, compared to the previously employed techniques, that only provided information regarding the average number of modified cysteine residues within PDI, not their exact identity. A combination of a phosphopeptide enrichment step and a MS-based approach was utilized to identify three phosphorylation sites on hYVH1, an atypical dual specificity phosphatase that functions as a cell survival factor. With the help of phosphomimetic and non-phosphorylable mutants, we were able to decipher their effect on localization and progression through the cell cycle. Collectively, these studies manifest the power of MS-generated data to influence and guide many different fields ranging from molecular embryology to biochemistry

Investigation of proteins and their modifications using high-resolution mass spectrometry

Advances in mass spectrometry (MS) has allowed for the deep analysis of various proteomes, providing identifications of proteins and their modifications. The true power in modern-day proteomics is the application of MS techniques to address various biological questions, propelling disease research and biochemical understanding of organisms. We have utilized high-resolution mass spectrometry to investigate biological questions leading to a greater knowledge of cellular biology. The transcriptional co-activator with PDZ-binding motif (TAZ), is regulated by reversible phosphorylation. However, sequence analysis suggests many potential uncharacterized sites of TAZ phosphorylation, specifically in regions in close proximity to a critical phosphorylation site making site assignment challenging. Using both targeted and untargeted approaches, we identified novel TAZ phosphorylation sites, using a reaction monitoring scheme to resolve positional phosphoisomers, and determined the biological consequence of a novel site, serine 93, on TAZ localization. Spinal muscular atrophy (SMA) is a motor neuron disease affecting 1 in 10,000 individuals. SMA has been shown to involve the release of extracellular vesicles (EVs), which have been used as a source of biomarkers for disease. We examined the use of EVs as a source of SMA biomarkers. We isolated and quantified \u3e650 proteins from SMA-derived vesicles finding potential biomarkers, one of which was confirmed in patients, suggesting these vesicles coupled with our methods are suitable for SMA biomarker discovery. In the model plant species Arabidopsis thaliana gene expression is heavily regulated through post-translational ubiquitination, however a gap between the number of ubiquitinated substrates identified and genes encoding the ubiquitin machinery exists, suggesting many unidentified modifications exist. The main strategy for studying ubiquitomes across species uses diglycine enrichment followed by MS analysis. We developed a DIA-based MS method coupled with novel sample preparation methods to overcome plant-specific challenges and increase the repository of the Arabidopsis ubiquitome, identifying 160 proteins with over 400 ubiquitination sites. The prevalent Charcot-Marie-Tooth disease can be caused by mutations in the lipid phosphatase MTMR2, a protein critical for regulating endosomal dynamics. MTMR2 is regulated by phosphorylation at serine 58. However, the phosphatase and the alterations in protein-protein interactions occurring with this modification have not been thoroughly investigated. To isolate MTMR2 interacting proteins, we utilized in vivo labeling fusing BirA biotin ligase to MTMR2, followed by MS analysis, identifying a putative interactor, TSSC1. We also provide evidence that MTMR2 itself may be subjected to phosphorylation-dependent degradation. This work utilizes high-resolution MS techniques to link protein regulation and function in a variety of biological and cellular contexts. The techniques presented here can be applied to address the gaps of knowledge in various proteomes and are amenable to user-specific modifications. The techniques here provide a framework for determining disease biomarkers for neurological diseases from EVs, investigating proteome-wide changes through protein modifications, and ultimately link high-resolution analytical mass spectrometry techniques and data to address critical biological events in a robust fashion

Regulation of genome stability and cell cycle progression by SUMOylation

Post translational modifications (PTMs) are orchestrated by highly active and reversible enzymatic systems to regulate the functional diversity of proteins. Because of their dynamic nature, PTMs are used by the cell as a controllable system to regulate a wide variety of processes. Studying modifications of proteins will give us more insight in how the cell uses PTMs to regulate cellular processes and how different PTMs act together to adjust the function of proteins. The research described in this thesis focuses one of these PTMs, the Small ubiquitin-Like Modifier (SUMO). SUMOs are proteins that are covalently attached to lysines in target proteins. These studies have uncovered hundreds of SUMO target proteins and acceptor sites and revealed a role for SUMOylation in protein degradation, cell cycle progression and DNA repairLUMC / Geneeskund

Discovery and Characterization of Methylation of Arginine 42 on Histone H3: A Novel Histone Modification with Positive Transcriptional Effects

Eukaryotic genomic DNA is packaged in the form of chromatin, which contains repeating nucleosomal units consisting of roughly two super-helical turns of DNA wrapped around an octamer of core histone proteins composed of four histone species: one histone H3/H4 tetramer and two histone H2A/H2B dimers. Histones are basic globular proteins rich in lysine and arginine residues, with unstructured N-terminal “tail” regions protruding outside the nucleosome structure, and structured “core” domains in the DNA-associated portion. Several core residues, and in particular arginines in H3 and H4, mediate key interactions between the histone octamer and DNA in forming the nucleosomal particle. Histone post-translational modifications (PTMs) lead to downstream effects indirectly by allowing or preventing docking of effector molecules, or directly by changing the intrinsic biophysical properties of local chromatin. To date, little has been done to study PTMs that lie outside of the unstructured tail domains of histones. I describe here the identification by mass spectrometry of a novel methylation site on histone H3, the asymmetric dimethylation of arginine 42 (H3R42me2a). H3R42 is conserved through evolution and is at the DNA entry/ exit position within the nucleosome core, with likely interactions with the DNA backbone. I show that methyltransferases CARM1 and PRMT6 methylate this residue in vitro and in vivo. Using chemically-defined “designer” histones I also show that methylation of H3R42 stimulates transcription in vitro from chromatinized templates. Using peptide pull down experiments combined with enzymatic assays I demonstrate that H3R42me2a prevents the stimulation of the histone deacetylase activity of the N-CoR co-repressive complex by impeding its binding to H3. Thus, H3R42 is a new histone methylation site with stimulating effects on transcription. I propose that methylation of basic histone residues at the DNA interface may be a general mechanism to disrupt histone:DNA interactions, with effects on downstream processes, including transcription

Proteomic snapshot of the EGF-induced ubiquitin network

In this work, the authors report the first proteome-wide analysis of EGF-regulated ubiquitination, revealing surprisingly pervasive growth factor-induced ubiquitination across a broad range of cellular systems and signaling pathways

Towards Understanding the Mechanism of Wolbachia-Induced Cytoplasmic Incompatibility

Wolbachia is a vertically transmitted intracellular bacterium that infects a large number of arthropods and filarial nematodes. Many strains of Wolbachia manipulate host reproduction through cytoplasmic incompatibility (CI). In its simplest form, CI is a phenomenon where male host infected with Wolbachia cannot produce viable offspring with uninfected females; this apparent sterility is restored when the male host mates with Wolbachia-infected females—a phenomenon called rescue. Since Wolbachia is transmitted through the female germline, CI helps Wolbachia propagate in the host population. These properties of CI have been utilized in Wolbachia-based methods to control mosquito-borne diseases such as Dengue and Zika and these methods have proven to be effective in field trials.In recent years, pairs of Wolbachia proteins expressed by two syntenic genes have been discovered as CI factors (Cifs) that is responsible for CI induction and rescue. They are able to recapitulate the CI-like defect and rescue phenomena when expressed in transgenic Drosophila, mosquito and yeast S. Cerevisiae. Different functional models have been proposed to explain how CI factors induce and rescue CI. However, the detailed molecular mechanism remains elusive. This thesis describes projects aimed at elucidating the mechanism of Wolbachia-induced CI. In the first two projects, we aim to identify the enzymatic target of CidB—a CI factor possessing deubiquitylase (DUB) activity. DUB is an enzyme that specifically cleaves ubiquitin, an important post-translational modifier protein, from its substrates. We first developed and characterized an unbiased tool based on OtUBD to purify ubiquitylated proteins from biological samples. Using OtUBD pulldowns and proteomics, we were able to screen for candidate DUB substrates of CidB in yeast. OtUBD also proves to be a versatile, efficient, and economical new tool for general ubiquitin-related research with specific advantages over other methods, such as in detecting monoubiquitylation or ubiquitin linkages to noncanonical sites. In another project, we investigated the role of the interactions between the cognate Cif pair—CinA and CinB—in CI rescue. Through collaboration, we solved the crystal structure of the CinA-CinB complex. With structurally guided mutagenesis, we demonstrated that the binding between CinA-CinB is crucial for rescue in yeast and transgenic Drosophila systems. These experiments provide evidences to differentiate between different CI models. In summary, the studies described here offer valuable insights towards understanding of the mechanism of CI as well as a novel method to study ubiquitin-related topics in general

Towards the absolute quantification of protein isoforms through the use of stable-isotope dilution mass spectrometry

While the existence of protein was first described by Berzelius and Mulder back in 1838 and a single empirical formula noted (C400H620N100O120P1S1) (Vickery, 1950, Brand, 1946), early protein-based research was limited to the analysis of proteins which could be easily purified in large quantities, such as those obtained from blood, egg whites and those obtainable from slaughterhouses, such as digestive and metabolic enzymes (Chapman, 2005). Indeed, despite the development of recombinant deoxyribonucleic acid technologies in the 1970s (enabling protein expression) and the increasing sensitivity of techniques which enable the identification and sequencing of proteins separated by gel electrophoresis (Patterson and Aebersold, 2003), it was not until the late 1980s, with the description of soft biomolecule ionisation that large scale proteomic analyses were undertaken, based upon the use of mass spectrometry (Guerrera and Kleiner, 2005). While early mass spectrometry-based proteomic analyses focussed on the systematic identification of a great number of proteins within a single organism, the field of proteomics is now becoming increasingly quantitative (Baak et al., 2005), enabling the relative comparison of protein expression patterns between phenotypes, but also the targeted absolute quantification of specific proteins. During this project, a stable isotopically labelled internal standard based absolute quantitative technique, first described by Gerber and co-workers in 2003 (S. A. Gerber et al., 2003), was applied to the absolute quantification of three families of multiple protein isoforms. This area of research is of particular scientific interest as it is thought that up to 95% of human multi-exon genes may be subject to alternative splicing, making alternative splicing the rule, not the exception (Pan et al., 2008a). Indeed alternative splicing has also been implicated as both a cause and a consequence of disease. This technique should therefore enable both the confirmation of disease, based upon the identification of a set of phenotype specific protein biomarkers, but also the mapping of a disease’s progression (Venables, 2004). During this study, stable isotopically labelled internal standard peptides were selected for the absolute quantification of 11 confirmed protein isoforms, and two predicted protein isoforms. In addition, a separate MRM based LC-MS acquisition method was developed for the absolute quantification of each of the three families of protein isoforms (A-Raf, PDE4B and SERCA2) within a single analysis, and finally, these acquisition methods were applied to the absolute quantification of a range of immunoprecipitated, exogenously expressed protein isoforms. This project was, however, hindered by the sensitivity of the mass spectrometers available for use, preventing these acquisition methods from being applied to the absolute quantification of the endogenous levels of protein expression. While beyond the scope of this project, the further development of this quantitative technique should enable future researchers to: (i) Quantify each endogenously expressed protein isoform within a family of multiple protein isoforms. (ii) Assess any changes in the expression of each isoform in a range of cellular states, and (iii) Assess how a targeted drug treatment may affect the expression ratio of these protein isoforms

Determinación de nuevas funciones de la quinasa DYRK2 en respuesta a estrés genotóxico

Cancer is a complex disease enhanced by alterations in signaling pathways. In fact, DNA damage response (DDR) pathways play a key role in cancer development when they are deregulated. DDR pathways induce a cellular response that includes principally DNA repair pathways activation, cell cycle arrest, and cell death. Although there is a great knowledge of the signaling pathways that take place in DDR, further research is required to assess new molecular mechanisms for fully comprehend this complex scheme. In this sense, kinases like DYRK2, with an important role in DDR, are key to better understand and control DNA genomic damage situations. Thus, identification of new substrates for this kinase, as well as to elucidate novel pathways implicated in DDR would open a road to the development of new therapeutic strategies against cancer. In the present work we show NOTCH1 as a novel substrate for DYRK2. We describe for the first time a new regulation mechanism of the NOTCH1 signaling pathway mediated by this kinase. We demonstrate that DYRK2 phosphorylates Notch1-IC in response to DNA damage and facilitates its proteasomal degradation by FBXW7 ubiquitin ligase through a Thr2512 phosphorylation-dependent mechanism. We show that DNA damage-dependently triggered DYRK2 has a relevant effect on the viability, motility and invasion capacity of cancer cells expressing NOTCH1. In summary, we reveal a novel DRYK2-dependent mechanism of regulation for NOTCH1 which might help us to better understand its role in cancer biology. Besides, we attempted to overview of cellular circuits that are involved in ionizing radiation triggered DNA damage response in human dermal fibroblasts. To allow this, we employed three different approaches: RNA-seq and proteomic and phosphoproteomic analysis based on SILAC labelling. This is, to our knowledge, the first work that compare these 3 different functional approaches to this aim. The description of NOTCH1 as a new DYRK2 substrate, as well as the acquisition of solid data compilation that would permit a deeper understanding of the pathways that take place in response to ionizing radiation. This will provide new therapeutic opportunities for radiodermatitis prevention and treatment. Taken together, these data contribute to better understand the network of signaling pathways activationdeactivation under DNA damage conditions, opening a road to the development of new therapeutic strategies against cancer.El cáncer es una enfermedad compleja que se ve impulsada por alteraciones en rutas de señalización. De hecho, las rutas de respuesta al daño al ADN juegan un papel esencial en el desarrollo de cáncer cuando se encuentran desreguladas. Estas rutas inducen una respuesta celular que incluye principalmente la reparación del ADN, el arresto del ciclo celular y la muerte celular. A pesar de que existe un amplio conocimiento de las rutas de señalización de respuesta al daño al ADN, una mayor investigación es necesaria para detectar nuevos mecanismos moleculares que nos permitan comprender este complejo entramado de señales. En este sentido, proteínas como DYRK2 son esenciales para entender y controlar situaciones de daño al ADN. Por tanto, la identificación de nuevos sustratos para esta quinasa, así como de nuevas rutas de señalización implicadas en la respuesta al daño al ADN podrían ser herramientas muy útiles en el desarrollo de estrategias contra esta enfermedad. En este trabajo mostramos cómo NOTCH1 es un nuevo sustrato de DYRK2. Describimos por primera vez un nuevo mecanismo regulatorio de la ruta de señalización de Notch por medio de esta quinasa. Demostramos que DYRK2 fosforila a NOTCH1-IC en la Thr2512 en respuesta a daño al ADN y promueve su degradación proteosomal gracias a la ubiquitinación por parte de la ubiquitín-ligasa FBXW7. Además, mostramos que el efecto de DYRK2 en respuesta a estrés genotóxico tiene un efecto relevante en la viabilidad, motilidad y capacidad de invasión de células tumorales que expresan NOTCH1. En resumen, mostramos un nuevo mecanismo de regulación de NOTCH1 por parte de DYRK2 que podría ayudarnos a comprender mejor su papel en la biología del cáncer. Además, hemos tratado de obtener una visión general de los circuitos celulares implicados en la respuesta al daño al ADN generado por radiación ionizante en fibroblastos. Para ello hemos analizado 3 niveles ómicos distintos: transcriptómico (por RNA-seq), proteómico y fosfoproteómico (empleando SILAC). Este sería hasta ahora el primer trabajo que emplea estas 3 aproximaciones diferentes para este fin. En resumen, en este trabajo se describe NOTCH1 como un nuevo sustrato de DYRK2, así como la adquisición de datos sólidos que permiten una comprensión más profunda de las rutas que se activan en respuesta al daño al ADN. En conjunto, estos data contribuyen a una mejor comprensión de las redes de señalización que se activan y desactivan en respuesta al daño al ADN, abriendo una puerta al desarrollo de nuevas estrategias terapéuticas frente al cáncer

Investigating Chemical Modifications in a Complex Proteome

Thesis advisor: Eranthie WeerapanaThesis advisor: Jianmin GaoProteins are composed of the 20 naturally occurring amino acids and are further modified by a variety of post-translational modifications (PTMS). Naturally occurring amino acids are diverse in structure and function. Catalytic amino acids, or nucleophilic amino acids, are of particular interest because of their contribution to chemical transformations in the cell. Synthetic covalent modification is a means to further functionalize or diversify proteins. These modifications, or enhancements, allow for improved understanding of protein structure, function and activity. For instance, isotope labeling of amino acid side chains in NMR studies enable investigators to study protein dynamics upon substrate or ligand binding. Fluorescence labeling is particularly useful to investigate protein cellular localization. Covalent modification is a useful tool to investigate the relative level of activity for protein known to be regulated by PTMs. An important feature of covalent modification reactions is site specificity, as this dictates the location, number of modifications, and protein targets. Tyrosine is of particular interest because it is both nucleophilic and aromatic. These characteristics contribute to the existence of tyrosine residues in both the protein surface and hydrophobic cores. Tyrosine is incorporated into proteins at a relatively low frequency. Unlike lysine, which is ubiquitous on protein surfaces, the low number of potential sites for general tyrosine modifications makes it an attractive site for surface bioconjugation modifications. A low number of surface modifications is less likely to perturb native protein function. Bioconjugation reactions give access to functionalizing the surface of proteins with moieties such as fluorophores, PEG, peptides, or drugs. Tyrosine is an attractive target for modifications because it is found in the active sites of a variety of enzymes such as sialidases, glutathione-S transferases, corticosteroid 11-beta-dehydrogensase, DNA topoisomerase, and ferredoxin-NADP+ reductase. Provided here is a survey of the known non-selective and selective synthetic chemical modification reactions for tyrosine. To investigate nucleophilic amino acids, Activity Based Protein Profiling (ABPP) may be implemented to investigate the role of these residues. ABPP utilizes small molecule covalent probes as a tool to selectively target enzymes in their active state. To investigate a protein of interest (POI) (or class of proteins) by ABPP, it is necessary to use a small molecule covalent probe that selectively reacts with the POI over other proteins within the proteome. Due to this requirement, it is necessary to expand the current ABPP probe toolbox to increase the coverage of what proteins in the proteome may be studied. Inspired by findings in the literature, our lab sought to explore the utility of various aryl halides for implementation in ABPP probes to overcome this limitation. This study revealed dichlorotriazine as a biologically relevant and reactive electrophile. A focus was placed on a dichlorotriazine containing probe library (LAS1-LAS20). LAS17 was discovered to be a potent and selective inhibitor of human glutathione S-transferase pi (GSTP1). Further studies revealed GSTP1 as a novel therapeutic target for the treatment of triple negative breast cancer. Other studies revealed several members of the dichlorotriazine library were found to covalently modify purified recombinant human aldolase A (ALDOA) in the presence of a complex cellular background. Additionally, LAS9 was identified as an inhibitor of ALDOA retro aldol condensation activity in vitro. Lastly, the final chapter highlights two collaborations in which tandem mass spectrometry experiments aid in the characterization of experimental data. In the first collaboration, a quantitative cysteine reactivity profiling method was used to characterize the selectivity of a cysteine reactive covalent NRF2-inducing small molecule, MIND4-17. In the second collaboration, analysis of tryptic mass spectrometry data enabled high resolution characterization of peptide sequencing for superfolder green fluorescent protein (sfGFP) expressed from observed internal nonsense suppression. Identification of the misincorporated amino acid facilitated the elucidation of the cross-talk mechanism.Thesis (PhD) — Boston College, 2017.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Chemistry