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 protein). 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 would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

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

TMPRSS2-ERG gene fusion, a molecular alteration found in nearly half of primary prostate cancer cases, has been intensively characterized at the transcript level. However limited studies have explored the molecular identity and function of the endogenous fusion at the protein level. Here, we developed immunoprecipitation-mass spectrometry assays for the measurement of a low-abundance T1E4 TMPRSS2-ERG fusion protein, its isoforms, and its interactome in VCaP prostate cancer cells. Our assays quantified total ERG (∼27,000 copies/cell) and its four unique isoforms and revealed that the T1E4-ERG isoform accounted for 52 ± 3% of the total ERG protein in VCaP cells, and 50 ± 11% in formalin-fixed paraffin-embedded prostate cancer tissues. For the first time, the N-terminal peptide (methionine-truncated and N-acetylated TASSSSDYGQTSK) unique for the T1/E4 fusion was identified. ERG interactome profiling with the C-terminal, but not the N-terminal, antibodies identified 29 proteins, including mutually exclusive BRG1- and BRM-associated canonical SWI/SNF chromatin remodeling complexes. Our sensitive and selective IP-SRM assays present alternative tools to quantify ERG and its isoforms in clinical samples, thus paving the way for development of more accurate diagnostics of prostate cancer.

Rational Design and Development of SARS-CoV-2 Serological Diagnostics by Immunoprecipitation-Targeted Proteomics.

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.

Mass spectrometry-based proteomics in basic and translational research of SARS-CoV-2 coronavirus and its emerging mutants.

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.