Biology subjects

Woolfitt, A. R.

Publications and source records attributed to Woolfitt, A. R..

3 recordsLinked to original sources

bioRxiv2023

Inclusion of glycopeptides in hydrogen/deuterium exchange mass spectrometry analysis of SARS-CoV-2 spike ectodomain provides increased sequence coverage

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide precise analysis of a proteins conformational dynamics across varied states, such as heat-denatured vs. 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 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. We report HDX-MS analysis of the SARS-CoV-2 spike protein ectodomain in its trimeric pre-fusion form, which has 22 predicted N-glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their isotopic mass shifts from deuteration. Inclusion of the deu-terated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural re-arrangements. Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/544985v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1d8e1f6org.highwire.dtl.DTLVardef@1db0774org.highwire.dtl.DTLVardef@c68d1eorg.highwire.dtl.DTLVardef@15aed98_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Comprehensive characterization of toxins defines progression and stages in a non-human primate model of inhalation anthrax

Inhalation anthrax has three clinical stages: early-prodromal, intermediate-progressive and late- fulminant. The toxins produced during infection exert pathologic effects leading to death, but they have not been comprehensively characterized throughout the course of infection. Mass spectrometry methods for anthrax toxins, total-protective antigen (PA), total-lethal factor (LF), total-edema factor (EF), and toxin complexes, lethal toxin and edema toxin were used to characterize the stages of inhalation anthrax in 23 cynomolgus macaques. The target aerosol dose was 200 LD50 B. anthracis Ames spores. 22 animals died during the study. Different patterns of toxemia and bacteremia were observed in 11 animals with the shortest survival times (fast progression), the 11 animals with longer survival (slow progression), and the one survivor. Toxemia and bacteremia were predominantly triphasic with an early rise (phase-1), a plateau/decline (phase-2), and a final rapid rise (phase-3). The patterns were consistent for all toxins. The end-of-phase-1 LF was higher in fast progression [median(lower quartile- upper quartile)] of [195(57.4-326)-ng/mL], than in slow progression [23.8(15.6-26.3)-ng/mL] (p=0.0001), or the surviving animal [11.1-ng/mL]. End-of-phase-1 EF was also higher in fast [22.2(2.7- 42.8)-ng/mL] than slow progression [0.17(0.064-0.066)-ng/mL] (p=0.0005), or the surviving animal [0.040-ng/mL]. Animals with slow progression and lower end-of-phase-1 toxemia, had an extended plateau/decline ([≥]24-hours), with low variability of PA, LF, and LTx across all animals. Its characterization revealed an upper threshold; a limit for exiting phase-2 and entering the critical phase-3, 342-ng/mL (PA), 35.8-ng/mL (LF), and 1.10-ng/mL (EF). The thresholds were exceeded early in animals with fast progression (38.3{+/-}7.4-hours) and later in slow progression (78.7{+/-}14.1-hours). Once the threshold was passed toxin levels rose rapidly, differences in toxemia were reduced, and the duration to terminal was rapid and similar; 21.0{+/-}7.3-hours for fast and 20.4{+/-}7.3-hours for slow. This first comprehensive evaluation of anthrax toxins defined all stages, providing insights into disease progression. Author SummaryThe comprehensive analysis of all major anthrax toxins and bacteremia in a non-human primate model of inhalation anthrax revealed a triphasic kinetics of toxemia that aligns with the three clinical stages, early-prodromal, intermediate-progressive and late-fulminant. End of phase-1 toxin levels may predict the subsequent speed of progression. Phase-2 toxemia helped define critical thresholds representing the entry to phase-3. Exceeding these thresholds was associated with a short remaining survival time (about 21 hours). This first comprehensive characterization of toxemia provides knowledge and guidance for better management of anthrax.

microbiology↗

bioRxiv2021

N-glycosylation profiles of the SARS-CoV-2 spike D614G mutant and its ancestral protein characterized by advanced mass spectrometry

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.

biochemistry↗