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Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire

ABSTRACT: Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including complete variable regions in Ig light and heavy chains. Ig-MS uses new advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and degree of clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without DNA/RNA sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Neil Kelleher

PROVIDER: MSV000087529 | MassIVE | Thu May 27 10:41:00 BST 2021

REPOSITORIES: MassIVE

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Next-generation Serology by Mass Spectrometry:Readout of the SARS-CoV-2 Antibody Repertoire

Project description:Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses new advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and degree of clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

2022-09-24 | PXD028627 | panorama

Divergent Antibody Repertoires Found for Omicron versus Wuhan SARS-CoV-2 Strains using Ig-MS

Project description:SARS-CoV-2 Omicron (B.1.1.529) and its subvariants are currently the most common variants of concern worldwide, featuring numerous mutations in the spike protein and elsewhere that collectively make Omicron more transmissible and more resistant to antibody-mediated neutralization provided by vaccination, previous infections, and monoclonal antibody therapies than its predecessors. We recently reported the creation and characterization of Ig-MS, a new mass spectrometry-based serology platform that can define the repertoire of antibodies against an antigen of interest at single proteoform resolution. Here, we applied Ig-MS to investigate the evolution of plasma antibody repertoires against the receptor-binding domain (RBD) of SARS-CoV-2 in response to the booster shot and natural viral infection. We also assessed the capacity for antibody repertoires generated in response to vaccination and/or infection with the Omicron variant to bind to both Wuhan- and Omicron-RBDs. Our results show that 1) the booster increases antibody titers against both Wuhan- and Omicron- RBDs and elicits an Omicron-specific response and 2) vaccination and infection act synergistically in generating anti-RBD antibody repertoires able to bind both Wuhan- and Omicron-RBDs with variant-specific antibodies.

2022-08-24 | MSV000090199 | MassIVE

Decoding the protein composition of whole nucleosomes with Nuc-MS

Project description:Nuc-MS characterizes histone modifications and variants directly from intact endogenous mono-nucleosomes. Preserving whole nucleosome particles enables precise interrogation of their protein content, as for H3.3-containing nucleosomes which had 6-fold co-enrichment of variant H2A.Z over bulk chromatin. Moreover, Nuc-MS showed co-occurrence of oncogenic H3.3K27M with euchromatic marks (e.g., H4K16ac and >40-fold enrichment of H3K79me2). By capturing the entire epigenetic landscape, Nuc-MS provides a new, quantitative readout of nucleosome-level biology.

2020-04-06 | MSV000085238 | MassIVE

Proliferative Receptor Binding Domain (RBD)-specific CD4+ T cells correlate with neutralizing antibody levels after natural SARS-CoV-2 infection and are induced following vaccination

Project description:Long-term immunity to SARS-CoV-2 infection, comprising neutralizing antibodies and T cell-mediated immunity, in a very large majority of the population, is required to reduce the current pandemic. We have investigated in detail the association between memory CD4 and CD8 T cells in recovered COVID-19 subjects, and their levels of neutralizing antibodies.The results show that RBD-specific memory CD4 T cells were particularly variable, and up to half of recovered individuals lacked these CD4 T cells and had much lower neutralizing antibody titres. Conversely, study of additional subjects identified as having low neutralizing antibody titres had very low levels of RBD-specific CD4 T cells. Furthermore, at both the protein and transcriptomic levels, these low antibody subjects had spike-specific CD4 T cells with a higher proportion of an inhibitory phenotype, including Foxp3 and CTLA-4, and less effector phenotype with T-bet and cytotoxic granzymes and perforin. Vaccination led to an improvement in RBD-specific memory CD4 T cells and neutralizing antibody titres in these at-risk subjects. Our results suggest that epitopes within the RBD-region should be the particular target of booster vaccines.

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Shotgun proteomic elucidation of Synergistic mechanisms of Cannabidiol and SN-38 in the MCF7 cells

Project description:We evaluted the synergyistic interactions of cannabidiol (CBD) with standard chemotherapeutic agents such as Docetaxel (DOC), doxorubicin (DOX), paclitaxel (PTX), vinorelbine (VIN), and 7-Ethyl-10-hydroxycamptothecin (SN38) in MCf7 cells ultising different synergy metrics. Then we explored the synergistic mechanisms on the proteome level of the most synergistic combination (CBD and SN38 in a molar ratio of 371:1, respectively)

2021-11-03 | PXD026587 | Pride

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Project description:Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the ‘histone code’. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

2021-04-20 | GSE149484 | GEO

A high throughput pipeline for antibody validation

Project description:Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we show that an inverted version of WB allows parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). The pipeline provides means for large-scale implementation of concepts proposed by an international working group on antibody validation (IWGAV).

2018-07-26 | PXD010510 | Pride

Characterizing adjuvants’ effects at the murine immunoglobulin repertoire level

Project description:We developed an improved library prep protocol and standardized the data analysis pipeline for accurate repertoire profiling. In addition, two metrics were implemented to assess repertoire clone properties. We then studied systemically the effects of two adjuvants, CpG and Alum, on the Ig heavy chain repertoire using the ovalbumin (OVA) challenged mouse model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in terms of sequence mutation frequency, IGHV gene usage, CDR3 length, rescaled Hill numbers for clonal diversity, and clone selection strength. As a result, Ig repertoires of different tissues or isotypes exhibited distinguishable profiles at the non-immunized steady state. Adjuvanted immunizations further resulted in statistically significant alterations in Ig repertoire compared with PBS or OVA alone immunized groups. Lastly, we applied unsupervised machine learning techniques – multiple factor analysis and clustering – to identify Ig repertoire signatures in different compartments and under varying immunizations.

2023-04-07 | GSE228856 | GEO

ChIP-seq for RFX1

2023-11-08 | GSE210668 | GEO

Analysis of E2F2 interacting proteins

Project description:In an attempt to identify proteins interacting with transcription factor E2F2, proteins extracted from HA-E2F2 overexpressing HEK293T cells were were subjected to immunoprecipitation (IP) with an anti-HA antibody or, as a negative control (NegCt), with an anti-T antibody. Immunoprecipitated proteins were eluted, digested with trypsin, and the resulting peptides were analyzed by LC-MS/MS. The whole procedure was repeated several times in an attempt to gauge reproducibility (nine replicates of the anti-HA IP and six replicates of the control IP).

2013-01-18 | PXD000026 | Pride

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