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Principal Investigator: Jason Williams
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2024
Award: $3,081,019
Funding agency: National Institute of Environmental Health Sciences
A variety of service and collaborative projects for mass spectrometry analyses have been or are being carried out within the Mass Spectrometry Research and Support Group with approximately 3000 samples analyzed from about 40 scientists representing about 25 principal investigators or core heads from 7 laboratory branches and the Division of Translational Toxicology.
One effort is in support of the protein expression function of the Structural Biology Core Laboratory and Dr. Bob Petrovich. The role of the MSRSG is to confirm gene expression at the protein level prior to the Structural Biology Core Laboratory handing materials over to their users.
We have also put effort into the development of lipidomics. This project involves the identification/characterization as well as the quantitation of compounds; thus, this project is included in both the identification/characterization project and the quantitation project. Extensive lipidomic analyses have been performed in collaboration with the laboratory of Dr. John Cidlowski.
In addition, mass spectrometry has been used to determine the extent of modification and the specific sites of modification on biomolecules. MS-based approaches have many advantages, including generally rapid analyses without radiolabeling. Protein characterization by mass spectrometry has been performed on a variety of proteins. Most of this work has been performed with electrospray mass spectrometry in conjunction with UPLC. Additionally, several proteins’ molecular architecture has been analyzed utilizing XL-MS techniques.
1. Proteomic analysis of IgA-protein aggregates. We are currently involved in a collaborative effort between the Division of Renal Diseases and Hypertension from the University of Minnesota at Minneapolis. IgA nephropathy is the most common form of glomerulonephritis worldwide. The hallmark of the disease is deposition of IgA1 in the glomerular mesangium. These deposited IgA1 are mainly polymeric in nature and include heteromeric complexes of IgA covalently bound to other plasma proteins. Identification of the key constituents of these protein-protein complexes may lead to better understanding of the pathophysiology of this disease. Approximately 10% of IgA1 in archival samples from IgAN patients and controls were found as high molecular mass complexes. Immunoblotting demonstrated 1:1 complex between IgA and albumin, alpha-1-antitrypsin, or alpha-1-microglobulin. Some complexes dissociated completely to free proteins and IgA1 when reducing agents were added, suggesting that these proteins were covalently linked through disulfide bonds (SS bonds). We were able to identify several inter-chain SS between IgA1 and other plasma proteins. A nonreducible thioether bridge, resulting of elimination of one of the sulfur atoms from the disulfide bond, was also identified in one of the heteromeric protein complexes.
2. Protein Crosslinking. Multiple projects have been analyzed to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. These experiments have been conducted using BS3 as the cross-linking reagent followed by trypsin digestion and nanoLC-ESI-MS performed on a Q-Exactive Plus mass spectrometer. While there have been successful analyses on multiple projects, we recently identified chemical crosslinks in the TSEN-CLP1 complex. In efforts to understand the role of the TSEN and tRNA splicing, multiple approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy. In addition, in collaboration with the P. Blackshear group, we have analyzed the chemical crosslinks in Gaboon caecilian TTP and the CNOT1 proteins. Additionally, in collaboration with the R.S. Williams group, where we have analyzed the intramolecular crosslinks within BS3 treated Senataxin. Finally, in collaboration with the W. Copeland group, we have analyzed the intra- and inter-molecular BS3 crosslinks between polymerase gamma and lonP protease.
3. Characterization of PTMs. In 2023 we collaborated with Ms. Carol Trempus and Dr. Stavros Garantziotis to investigate the phosphorylation of proteins from pdgfr-alpha positive fibroblasts isolated from murine lungs after PBS or bleomycin treatment. We used a relative quantification approach to evaluate changes in phosphorylation of proteins in the samples with the hopes that changes in the phosphorylation events might shed light on what signal transduction pathways were altered by bleomycin treatment. We are also currently collaborating with Dr. Carlos Guardia in the analyses of glycosylation and other PTMs on Autophagy-Related Protein 9A and 9B. Moreover, in collaboration with Drs. Anirban Kar and Paul Doestch, we have been analyzing the in vitro acetylation of NTHL1. Acetylation on DNA repair proteins is a dynamic epigenic modification regulated by acetyltransferases and lysine deacetylases. Accumulating evidence has indicated that the aberrant acetylation of DNA repair proteins contributes to the pathogenesis of cancer. Hence, probing the acetylation states of DNA repair proteins might be of great help in better understanding pathogenesis of cancer. NTHL1 is a bifunctional glycosylase involved in base excision repair. We were asked to identify the acetylation sites on four truncated variants of NTHL1 that were overexpressed as fusions to the C-terminus of bacterial MBP. The MBP-NHTL1 fusion proteins were subjected to in vitro acetylation using p300 protein as acetyl transferase before running the protein on an SDS-PAGE gel. Proteins in the gel bands were then reduced, alkylated, and digested with trypsin. Tryptic digests were analyzed using UPLC coupled to an orbitrap high resolution mass spectrometer. Nine lysine acetylation sites were identified in the four types of NHTL-1 constructs whereas 26 sites were detected on MBP. There were no significant differences in lysine acetylation profiles between p300-treated variants and the corresponding non-acetylated control. Additionally, the MSRSG has been collaborating with the laboratory of Dr. Robin Stanley to analyze the sites of attachment for various small molecule inhibitors of the Nsp15 polyuridylase from SARS-CoV-2.
Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><A1PI><Acetylation><Acetyltransferase><Affect><Albumins><Amino Acids><Architecture><Autophagocytosis><Base Excision Repairs><Berger's Disease><Bifunctional Reagents><Binding><Bleo><Bleomycin><Bright Disease><CD140a Antigens><COVID-19 virus><COVID19 virus><Cancers><Cellular biology><Chemicals><Chimera Protein><Chimeric Proteins><CoV-2><CoV2><Collaborations><Complex><Coupled><Cross-Linking Reagents><Crosslinking Reagents><Cryo-electron Microscopy><Cryoelectron Microscopy><DNA Base Excision Repair><DNA Repair Gene><DNA repair protein><Deacetylase><Deposit><Deposition><Development><Digestion><Disease><Disorder><Dissociation><Dysfunction><E1A Binding Protein p300><E1A-associated p300 protein><EP300><EP300 gene><Electron Cryomicroscopy><Elements><Engineering / Architecture><Enzyme Gene><Enzymes><Esteroproteases><Evaluation><Event><Fibroblasts><Functional disorder><Fusion Protein><Gel><Gene Expression><Glomerular Mesangium><Glomerular Mesangiums><Glomerulonephritis><Heymann Nephritis><Hypertensive Nephropathy><IGA Glomerulonephritis><IGA Nephropathy><IgA><IgA1><Immunoblotting><Immunoglobulin A><In Vitro><Intramural Research><KAT3B><Kidney Diseases><L-Lysine><Label><Laboratories><Life Cycle><Life Cycle Stages><Link><Lung><Lung Respiratory System><Lysine><Macromolecular Structure><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Metabolic Glycosylation><Methylation><Mice><Mice Mammals><Minnesota><Modification><Molecular><Molecular Interaction><Molecular Structure><Murine><Mus><Nature><Nephropathy><Normal Cell><PDGF alpha Receptor><PDGF receptor α><PDGF-R-alpha><PDGFR-α><PDGFRα><Pathogenesis><Patients><Peptidases><Peptide Hydrolases><Peptides><Phosphorylation><Physiopathology><Plasma Proteins><Platelet-Derived Growth Factor Receptor Alpha Polypeptide><Platelet-Derived Growth Factor alpha Receptor><Polymerase><Polymers><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Preparation><Prevention><Principal Investigator><Protease Gene><Proteases><Protein Modification><Protein Phosphorylation><Proteinases><Proteins><Proteolytic Enzymes><Proteome><Proteomics><R-Series Research Projects><R01 Mechanism><R01 Program><RNA Splicing><Radiolabeled><Reducing Agents><Reductants><Renal Disease><Renal Hypertension><Research><Research Grants><Research Project Grants><Research Projects><Resolution><Robin><Robin bird><Role><S element><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sampling><Scientist><Self-Help Groups><Services><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Side><Signal Transduction Pathway><Site><Splicing><Structure><Structure of glomerular mesangium><Sulfur><Support Groups><System><TC4 Protein><Techniques><Toxicology><Transfer RNA><Transferase><Transferase Gene><Tripcellim><Triplet Codon-Amino Acid Adaptor><Trypsin><Ubiquitilation><Ubiquitination><Ubiquitinoylation><Universities><Variant><Variation><Western Blotting><Western Immunoblotting><Work><Wuhan coronavirus><alpha 1 Antiprotease><alpha 1-Antiproteinase><alpha 1-Antitrypsin><alpha 1-Antitrypsin Trypsin Inhibitor><alpha 1-Protease Inhibitor><alpha 1-Proteinase Inhibitor><alpha(1)-microglycoprotein><alpha-1-microglobulin><aminoacid><autophagy><cell biology><coronavirus disease 2019 virus><coronavirus disease-19 virus><crosslink><cryo-EM><cryoEM><cryogenic electron microscopy><developmental><disulfide bond><experiment><experimental research><experimental study><experiments><focal glomerulonephritis><glycosylation><hCoV19><histone acetyltransferase p300><hypertensive kidney><immunoglobulin A glomerulonephritis><immunoglobulin A nephropathy><insight><insoluble aggregate><kidney disorder><life course><lipidomics><malignancy><mass spectrometer><mesangium><molecular mass><nCoV2><neoplasm/cancer><overexpress><overexpression><p300><p300 E1A-associated coactivator><p300 protein><p300-CBP coactivator><p300/CBP proteins><pathophysiology><platelet-derived growth factor receptor α><polymer><polymeric><preparations><protein HC><protein aggregate><protein aggregation><protein blotting><protein complex><protein crosslink><protein expression><protein structure><protein structures><proteins structure><pulmonary><radiolabeling><radiologically labeled><ran GTP-Binding Protein><ran Protein><ras-Related Nuclear Protein><renal disorder><resolutions><segmental glomerulonephritis><self help organization><small molecular inhibitor><small molecule><small molecule inhibitor><social role><structural biology><tRNA><thioether><transfer Ribonucleic acids><ubiquination><ubiquitin conjugation><α1-Antitrypsin><α1-Proteinase Inhibitor><α1-microglobulin>