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Principal Investigator: Leesa Deterding
Organization: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
Fiscal Year: 2021
Award: $574,792
Funding agency: National Institute of Environmental Health Sciences
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. The MS analysis of a variety of proteins has been investigated using mass spectrometry. Products and digests have been analyzed by both positive and negative ion MALDI mass spectrometry and LC in combination with electrospray mass spectrometry. In addition, we are currently analyzing the use of the crosslinker BS3 with a variety of proteins.
1. Glycosylation Characterization Studies. We are currently performing two projects related to glycosylation characterization. One project involves a recombinant SARS-CoV-2 spike glycoprotein which has recently been successfully expressed by the Structural Biology Core Laboratory at NIEHS. We have been asked to characterize the glycan structures and the degree of sequon occupancy on the glycoprotein. Our preliminary data indicate that all the sites on the recombinant protein are fully occupied and we are currently investigating the distribution of various glycoforms on each of 22 glycan sites by high resolution mass spectrometry. The other project involves an extramural collaborative effort between the Division of Renal Diseases and Hypertension from the University of Minnesota at Minneapolis and our group at NIEHS. Abnormalities in O-glycosylation of circulating IgA1 are implicated in the pathogenesis of IgAN. This was initially demonstrated by the altered binding of lectins with specificity for O-linked glycans and confirmed later by mass spectrometry. We aim to identify potential sources of discrepancy between the two analytical methods.
2. Histone Proteins. We have been collaborating with the Archer laboratory in an effort to examine the role of H1 in controlling gene expression and protein levels when we knock out the H1 gene. Knockout cell lines of the histone protein H1.4 protein (as it was determined by MS previously that it was phosphorylated) was compared to a "wild-type" cell line. Data were acquired and are being analyzed to determine if other histone levels change and/or their PTMS change if the H1.4 isoform is knocked out.
Also in collaboration with the Archer laboratory, the MSRSG evaluated changes in core and linker histone marks associated with proteosome inhibition. 13 histones were identified in these studies and 177 histone marks were identified in the histone H1 variants, consisting of 5 types of histone modifications: arginine methylation, lysine methylation, lysine acetylation, serine phosphorylation, and threonine phosphorylation. More than 133 PTMs-containing peptides were quantified and statistical testing using ANOVA and Tukeys HSD post-hoc test showed that very few histone marks were significantly changed.
3. Protein Crosslinking. Multiple projects are underway to characterize protein complexes by mass spectrometry in conjunction with chemical cross-linking. Most of 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, most recently we have determined the crosslinks in the multimeric Rix7 protein complex. In efforts to understand the role of the N-terminal domain in the Rix7 structure and this domains contribution to the proteins function, multiple approaches were used including chemical cross-linking and mass spectrometry as well as cryo-electron microscopy.
4. Characterization of RNA cleavage events. In collaboration with the Stanley lab at the NIEHS the MSRSG has characterized the RNA cleavage specificity for the NSP15 endoribonuclease from SARS-CoV-2. This work also characterized the nature of the resulting 3 ends of the cleavage products.
Other smaller projects include characterization of stable domains, post translational modifications, and extent of chemical labeling for various intramural research groups (Hall, Shaw, S. Williams, Wilson).
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><ANOVA><Acetylation><Affect><Amino Acids><Analysis of Variance><Arginine><Bifunctional Reagents><Binding><COVID-19 S protein><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID19 S protein><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><Cell Line><CellLine><Cellular biology><Chemicals><CoV-2><CoV2><Collaborations><Cross-Linking Reagents><Crosslinker><Crosslinking Reagents><Cryo-electron Microscopy><Cryoelectron Microscopy><Data><Digestion><Disease><Disorder><EXTMR><Electron Cryomicroscopy><Endoribonucleases><Enzyme Gene><Enzymes><Evaluation><Event><Extramural><Extramural Activities><Gene Expression><Gene Proteins><Genes><Glycans><Glycoproteins><Histone H1><Histones><Hypertensive Nephropathy><IgA1><Intramural Research><Ions><Isoforms><Kidney Diseases><Knock-out><Knockout><L-Arginine><L-Lysine><L-Serine><L-Threonine><Label><Laboratories><Lectin><Life Cycle><Life Cycle Stages><Link><Lipids><Lysine><MALD-MS><MALDI><MALDI-MS><Macromolecular Structure><Macropain><Macroxyproteinase><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Metabolic Glycosylation><Methylation><Minnesota><Modification><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Molecular Structure><Multicatalytic Proteinase><N-terminal><NH2-terminal><NIEHS><National Institute of Environmental Health Sciences><Nature><Nephropathy><Non-Polyadenylated RNA><Normal Cell><Pathogenesis><Peptides><Phosphorylation><Polysaccharides><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Prevention><Prosome><Proteasome><Proteasome Endopeptidase Complex><Protein Gene Products><Protein Isoforms><Protein Modification><Protein Phosphorylation><Proteins><Proteome><Proteosome><RNA><RNA Gene Products><RNA endonuclease><Radiolabeled><Recombinant Proteins><Recombinants><Renal Disease><Renal Hypertension><Resolution><Ribonucleic Acid><Role><SARS corona virus 2><SARS-CoV-2><SARS-CoV-2 S protein><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Safety><Sampling><Serine><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 corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Side><Site><Source><Specificity><Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization><Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization><Strains Cell Lines><Structure><System><Testing><Threonine><Tripcellim><Trypsin><Ubiquitilation><Ubiquitination><Ubiquitinoylation><Universities><Variance Analyses><Variant><Variation><Work><Wuhan coronavirus><aminoacid><analytical method><base><cell biology><cell type><conformation><conformational state><coronavirus disease 2019 S protein><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><cross-link><crosslink><cryo-EM><cryoEM><cultured cell line><experiment><experimental research><experimental study><functional group><glycosylation><hCoV19><histone modification><hypertensive kidney><insight><kidney disorder><life course><mass spectrometer><matrix assisted laser desorption ionization><multicatalytic endopeptidase complex><nCoV2><protein complex><protein crosslink><protein function><renal disorder><social role><structural biology><ubiquination><ubiquitin conjugation>