Current design of serological tests utilizes conservative immunoassay approaches and is focused on fast and convenient assay development, throughput, straightforward measurements, and affordability. Limitations of common serological assays include semiquantitative measurements, cross-reactivity, lack of reference standards, and no differentiation between human immunoglobulin subclasses. In this study, we suggested that a combination of immunoaffinity enrichments with targeted proteomics would enable rational design and development of serological assays of infectious diseases, such as COVID-19. Immunoprecipitation-targeted proteomic assays allowed for sensitive and specific measurements of NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum and enabled differential quantification of anti-SARS-CoV-2 antibody isotypes (IgG, IgA, IgM, IgD, and IgE) and individual subclasses (IgG1-4 and IgA1-2) in plasma and saliva. Simultaneous evaluation of the numerous antigen–antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic performance. Further validation revealed that anti-RBD IgG1, IgG3, IgM, and IgA1 levels were significantly elevated in convalescent plasma, while IgG2, IgG4, and IgA2 were not informative. Anti-RBD IgG1 levels in convalescent (2138 ng/mL) vs negative (95 ng/mL) plasma revealed 385 ng/mL as a cutoff to detect COVID-19 convalescent plasma. Immunoprecipitation-targeted proteomic assays will facilitate improvement and standardization of the existing serological tests, enable rational design of novel tests, and offer tools for the comprehensive investigation of immunoglobulin subclass cooperation in immune response.

b3 b4 b5 b8 b11 y2 y3 y4 y5 y7 y8 y9 y10 y11 y12 b12 c6 a7 N-term modifications Isoform abundance T1E4-ERG

Molecular diagnostics of the coronavirus disease of 2019 (COVID-19) now mainly relies on the measurements of viral RNA by RT-PCR, or detection of anti-viral antibodies by immunoassays. In this review, we discussed the perspectives of mass spectrometry-based proteomics as an analytical technique to identify and quantify proteins of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and to enable basic research and clinical studies on COVID-19. While RT-PCR and RNA sequencing are indisputably powerful techniques for the detection of SARS-CoV-2 and identification of the emerging mutations, proteomics may provide confirmatory diagnostic information and complimentary biological knowledge on protein abundance, post-translational modifications, protein–protein interactions, and the functional impact of the emerging mutations. Pending advances in sensitivity and throughput of mass spectrometry and liquid chromatography, shotgun and targeted proteomic assays may find their niche for the differential quantification of viral proteins in clinical and environmental samples. Targeted proteomic assays in combination with immunoaffinity enrichments also provide orthogonal tools to evaluate cross-reactivity of serology tests and facilitate development of tests with the nearly perfect diagnostic specificity, this enabling reliable testing of broader populations for the acquired immunity. The coronavirus pandemic of 2019–2021 is another reminder that the future global pandemics may be inevitable, but their impact could be mitigated with the novel tools and assays, such as mass spectrometry-based proteomics, to enable continuous monitoring of emerging viruses, and to facilitate rapid response to novel infectious diseases.

No abstract

SummaryTMPRSS2-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.