Last data update: Jun 24, 2024. (Total: 47078 publications since 2009)
Records 1-5 (of 5 Records) |
Query Trace: Keppel T [original query] |
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Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike glycoprotein
Haynes CA , Keppel TR , Mekonnen B , Osman SH , Zhou Y , Woolfitt AR , Baudys J , Barr JR , Wang D . Rapid Commun Mass Spectrom 2024 38 (5) ![]() ![]() Rationale: Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat-denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX-MS, but one challenge for complete sequence coverage is N-glycosylation sites. The deuteration of peptides post-translationally modified by asparagine-bound glycans (glycopeptides) has not always been identified in previous reports of HDX-MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data-dependent acquisition. An MS scan was used to identify precursor ions; if high-energy collision-induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low-energy collision-induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX-MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O. Results: We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Conclusion: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. |
N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry (preprint)
Wang D , Zhou B , Keppel TR , Solano M , Baudys J , Goldstein J , Finn MG , Fan X , Chapman AP , Bundy JL , Woolfitt AR , Osman SH , Pirkle JL , Wentworth DE , Barr JR . bioRxiv 2021 2021.07.26.453787 N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.Competing Interest StatementThe authors have declared no competing interest. |
N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry.
Wang D , Zhou B , Keppel TR , Solano M , Baudys J , Goldstein J , Finn MG , Fan X , Chapman AP , Bundy JL , Woolfitt AR , Osman SH , Pirkle JL , Wentworth DE , Barr JR . Sci Rep 2021 11 (1) 23561 ![]() N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins. |
Comprehensive Analysis of the Glycan Complement of SARS-CoV-2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.
Wang D , Baudys J , Bundy JL , Solano M , Keppel T , Barr JR . Anal Chem 2020 92 (21) 14730-14739 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic of coronavirus disease 2019 (COVID-19). The spike protein expressed on the surface of this virus is highly glycosylated and plays an essential role during the process of infection. We conducted a comprehensive mass spectrometric analysis of the N-glycosylation profiles of the SARS-CoV-2 spike proteins using signature ions-triggered electron-transfer/higher-energy collision dissociation (EThcD) mass spectrometry. The patterns of N-glycosylation within the recombinant ectodomain and S1 subunit of the SARS-CoV-2 spike protein were characterized using this approach. Significant variations were observed in the distribution of glycan types as well as the specific individual glycans on the modification sites of the ectodomain and subunit proteins. The relative abundance of sialylated glycans in the S1 subunit compared to the full-length protein could indicate differences in the global structure and function of these two species. In addition, we compared N-glycan profiles of the recombinant spike proteins produced from different expression systems, including human embryonic kidney (HEK 293) cells and Spodoptera frugiperda (SF9) insect cells. These results provide useful information for the study of the interactions of SARS-CoV-2 viral proteins and for the development of effective vaccines and therapeutics. |
Is there progress toward eliminating racial/ethnic disparities in the leading causes of death?
Keppel KG , Pearcy JN , Heron MP . Public Health Rep 2010 125 (5) 689-97 OBJECTIVES: We examined changes in relative disparities between racial/ethnic populations for the five leading causes of death in the United States from 1990 to 2006. METHODS: The study was based on age-adjusted death rates for four racial/ethnic populations from 1990-1998 and 1999-2006. We compared the percent change in death rates over time between racial/ethnic populations to assess changes in relative differences. We also computed an index of disparity to assess changes in disparities relative to the most favorable group rate. RESULTS: Except for stroke deaths from 1990 to 1998, relative disparities among racial/ethnic populations did not decline between 1990 and 2006. Disparities among racial/ethnic populations increased for heart disease deaths from 1999 to 2006, for chronic obstructive pulmonary disease deaths from 1990 to 1998, and for chronic lower respiratory disease deaths from 1999 to 2006. CONCLUSIONS: Deaths rates for the leading causes of death are generally declining; however, relative differences between racial/ethnic groups are not declining. The lack of reduction in relative differences indicates that little progress is being made toward the elimination of racial/ethnic disparities. |
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