Biology subjects

Drabovich, A. P.

Publications and source records attributed to Drabovich, A. P..

8 recordsLinked to original sources

bioRxiv2026

Proteome-Wide Serology Identifies Secreted IgD against RSV Fusion Glycoprotein as a Signature of Broadly Elevated Antibody Response

Serological evaluation of antibody response using conventional indirect immunoassays cannot capture the full depth, diversity, and functional complexity of human polyclonal antibodies. Here, we developed mass spectrometry-based Proteome-Wide Serology (ProWiSe) assays for the identification and absolute quantification of antigen-specific human antibodies across isotypes, subclasses, and variable-region repertoires. Using ProWiSe, we measured the depth and diversity of serum antibodies to multiple antigens of respiratory syncytial virus (RSV), a pathogen associated with repeated reinfections and skewed immune response. IgG1 antibodies targeting the fusion glycoprotein F (RSVF) dominated the antibody response, with a background-corrected median level of 1,700 ng/mL across 214 healthy maternal serum samples from the general population. ProWiSe identified reproducible co-enrichment of complement component 1q complexes, revealed the usage of several dozen IGHV variable-region germline genes in the anti-RSVF polyclonal response, and suggested limited IGHV repertoire diversity in some individuals. In addition to IgG1, we discovered a complex antibody response characterized by significantly elevated levels of nearly all human immunoglobulin isotypes and subclasses, including previously unexplored IgG4 and IgD. The identification of high-affinity IgD against prefusion and postfusion RSVF, but not against other antigens studied, was the most unexpected observation, occurring in 52% of the healthy maternal serum samples. ProWiSe unambiguously identified the secreted IgD isoform P01880-1 and supported the IgD paradox of preferential {lambda} light-chain usage in secreted IgD. Detectable anti-RSVF IgD levels ([≥]0.11 ng/mL) correlated with broadly elevated antibody responses across all other isotypes and subclasses, except IgE. ProWiSe assays offer a versatile platform for quantitative serological diagnostics, exploration of depth, diversity, and dynamics of polyclonal antibody response, and discovery of rare antibody isotypes.

immunology↗

bioRxiv2026

The Q226H Mutation in Avian H5N1 Hemagglutinin Mediates a Path towards Structural Adaptation in Humans

The global outbreak of highly pathogenic avian influenza (HPAI) A(H5N1) among birds and the spillover to mammals increases the risk for humans. A recent case in British Columbia with a clade 2.3.4.4b H5 virus infection revealed a mixture of 226Q/H in the receptor-binding site of hemagglutinin. While significant changes in pre-existing immunity by H1 or H3 polyclonal sera are not evident, we show that the Q226H mutation enables binding to human-type a2-6 sialic acid receptors. High-resolution cryo-EM structures provide a basis for the alteration in receptor preference and show that a possible path towards human adaptation also requires a conformational change of the bound a2-6-sialylated glycan. Continued surveillance for additional mutations that could enhance this phenotype is warranted.

immunology↗

bioRxiv2024

Identification and quantification of human relaxin proteins by immunoaffinity-mass spectrometry

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein), and a broader tissue specificity RLN2 (REL2_HUMAN). Due to their structural similarity, REL1 and REL2 proteins were collectively named a human relaxin protein in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays could reveal the identity and concentration of REL1 and REL2 in biological samples and facilitate evaluation of these proteins for diagnostic applications. RT-PCR revealed the high levels of RLN1 and RLN2 transcripts in prostate and breast cancer cell lines. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in numerous biological samples. IA-SRM assay of REL2 revealed its undetectable levels (<9 pg/mL) in control female and male sera, relatively high levels of REL2 in maternal sera (median 331 pg/mL, 120 patients), and a biphasic expression of REL2 across the gestational weeks. IA-SRM assays discovered potential cross-reactivity and false-positive measurements for relaxin immunoassays. The developed IA-SRM assays will facilitate investigation of physiological and pathological roles of REL1 and REL2 peptide hormones.

biochemistry↗

bioRxiv2023

The proteomic toolbox for identification, quantification, and characterization of polyclonal antibodies

Recent advances in proteomics and mass spectrometry facilitated the in-depth characterization of monoclonal antibodies and enabled innovative approaches for the quantification of polyclonal antibodies generated against numerous antigens. Human respiratory syncytial virus (RSV) is a contagious respiratory pathogen often manifested as a common cold infection in adults and more serious symptoms in infants and the elderly population. Here, we used a reference IgG1{kappa} monoclonal antibody NISTmAb 8671 and its affinity interaction with an RSV fusion glycoprotein F as a model to develop the proteomic toolbox for identification, quantification, and characterization of polyclonal antibodies. Our toolbox integrated a variety of proteomic and mass spectrometry approaches for accurate mass measurements of antibody fragments, antibody digestion with the complimentary proteases (trypsin, asparaginase, and proalanase), immunoaffinity enrichments, and bottom-up or middle-down proteomics. We measured absolute concentrations of anti-RSV antibody isotypes and subclasses in 69 serum samples of healthy individuals and revealed IgG1 (2,580 ng/mL), IgA1 (280 ng/mL), and IgM (180 ng/mL) as the most abundant isotypes. Interestingly, we also identified the presence of IgG2 (74 ng/ml), IgG4 (4.9 ng/mL) and IgA2 (5.5 ng/mL) antibodies. Interactome measurements detected the consistent co-precipitation of C1q complement complexes. Repertoire profiling of the variable regions of polyclonal antibodies revealed the frequent use of IGHV3 subgroup genes, while IGHV5-51 was the most abundant single gene of the anti-RSV polyclonal antibody response. The presented toolbox will facilitate the in-depth characterization of polyclonal antibodies and pave the way to quantitative approaches in serological studies and precision immunology.

biochemistry↗

bioRxiv2020

Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein with Orthogonal Immunoprecipitation-Mass Spectrometry Assays

TMPRSS2-ERG gene fusion, a molecular alteration driving nearly a half of prostate cancer cases, has been intensively characterized at the transcript level, while limited studies explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed and applied immunoprecipitation-mass spectrometry (IP-MS) assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms and its interactome in VCaP prostate cancer cells. IP-MS assays quantified total ERG ([~]27,000 copies/cell) and its four unique isoforms, and revealed that the T1E4-ERG isoform accounts for 71% of the total ERG protein in VCaP cells. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified and quantified. IP-MS with the C-terminal antibodies identified 29 proteins in the ERG interactome, including SWI/SNF chromatin remodeling complex subunits and numerous transcriptional co-regulators. Our data also suggested that TMPRSS2-ERG protein-protein interactions were exerted through at least two different regions. Knowledge on the distinct TMPRSS2-ERG protein isoforms and interactomes may facilitate development of more accurate diagnostics and targeted therapeutics of prostate cancer.

molecular biology↗

bioRxiv2018

Discovery of a human testis-specific protein complex TEX101-DPEP3 and selection of its disrupting antibodies

TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 antibody T4 disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.\n\nNon-standard abbreviations

molecular biology↗

bioRxiv2018

Identification of TEX101 functional interactome through proteomic measurement of human spermatozoa homozygous for the missense variant rs35033974

TEX101 is a testis-specific cell-surface protein expressed exclusively in the male germ cells and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility, and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm-oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 healthy fertile men and sub-fertile patients for a common and potentially deleterious missense variant rs35033974 of TEX101, and identified 52 heterozygous and 4 homozygous patients. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with near-complete degradation (>97%) of the corresponding G99V TEX101 form, and suggested that spermatozoa of homozygous patients could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified 8 cell-surface and 9 secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm-oocyte fusion (including LY6K and ADAM29) were confirmed by targeted proteomics and western blotting assays. Since recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a single pathogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found 5-fold lower (P=0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction.\n\nNon-standard abbreviations

molecular biology↗

bioRxiv2017

Multi-omics Biomarker Pipeline Reveals Elevated Levels Of Protein-Glutamine Gamma-Glutamyltransferase 4 In Seminal Plasma Of Patients With Prostate Cancer

PurposeProstate-specific antigen, a blood serum biomarker of prostate cancer, lacks specificity and prognostic significance, so considerable efforts are devoted to developing novel biomarkers. Seminal plasma, due to its proximity to prostate, is a promising fluid for biomarker discovery and non-invasive diagnostics. In this study, we investigated if seminal plasma proteins could increase specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers.\n\nExperimental DesignTo select 148 most promising biomarker candidates, we combined proteins identified through five independent data mining or experimental approaches: tissue transcriptomics, seminal plasma proteomics, cell secretomics, tissue specificity and androgen regulation. A rigorous biomarker development pipeline based on targeted proteomics assays was designed to evaluate the most promising candidates.\n\nResultsWe qualified 77 and verified 19 proteins in seminal plasma of 67 negative biopsy and 155 prostate cancer patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which could predict prostate cancer on biopsy and outperformed age and serum PSA. Machine-learning approaches also revealed improved multi-marker combinations for diagnosis and prognosis. In the independent verification set measured by an inhouse ELISA, TGM4 was up-regulated 3.7-fold (P=0.006) and revealed AUC 0.66 for detecting prostate cancer on biopsy for patients with serum PSA[≥]4 ng/mL and age[≥]50. Low levels of TGM4 (median 120 pg/mL) were detected in blood serum, but could not differentiate between negative biopsy, prostate cancer or prostate inflammation.\n\nConclusionsPerformance of TGM4 warrants its further investigation within the distinct genomic subtypes and evaluation for the inclusion into emerging multi-biomarker panels.

cancer biology↗