Protein Biomarker Discovery

Drabovich, Andrei P.; Martínez‐Morillo, Eduardo; Diamandis, Eleftherios P.

doi:10.1002/9781119081661.ch3

Cited by 9 publications

(7 citation statements)

References 122 publications

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“…MS has previously been used for the identification and quantification of the SARS-CoV-2 proteins in clinical samples. − Since systematic discovery and development of protein biomarkers involves numerous stages of verification and validation, MS assays due to their rapid design and execution are particularly useful at the early stages of biomarker discovery and development of diagnostic assays. − Without extensive fractionation, however, MS assays present relatively poor analytical sensitivity. Here, we suggested that a combination of immunoaffinity enrichment with MS measurements would provide sensitive and selective serological assays to measure viral proteins and antiviral immunoglobulins.…”

Section: Discussionmentioning

confidence: 99%

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

Rais

Dara

et al. 2022

Anal. Chem.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Rais

Dara

et al. 2022

Anal. Chem.

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“…Our approach for selection of the most promising testis-and germ cell-specific proteins relied on re-analysis of our previously identified spermatozoa (30) and seminal plasma proteomes (33), and on mining of the well-established proteomic databases. Since the high-quality protein assays are essential for biomarker discovery and development (45)(46)(47), we suggested that targeted proteomic assays due to their rapid design, development and execution could be particularly valuable tools to evaluate clinical utility of proteins lacking high-quality immunoassays or antibodies. We previously demonstrated that targeted proteomic assays provided robust quantification of proteins in human cell lines (48,49), primary cells (50,51), tissues (38), biological fluids (52)(53)(54)(55)(56), and serum (57)(58)(59).…”

Section: Discussionmentioning

confidence: 99%

Germ cell-specific proteins ACRV1 and AKAP4 facilitate identification of rare spermatozoa in semen of non-obstructive azoospermia patients

Zhang

Kanoatov

Jarvi

et al. 2022

Preprint

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Non-obstructive azoospermia (NOA), the most severe form of male infertility due to testicular failure, could be treated with intra-cytoplasmic sperm injection (ICSI), providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). Here, we hypothesized that some testis- and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining and extensive verification by targeted proteomic assays and immunofluorescent microscopy revealed a panel of testis-specific proteins expressed at the continuum of germ cell differentiation, including the late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) with the exclusive expression in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay revealed low numbers of the morphologically intact AKAP4+/ACRV1+/Hoechst+ spermatozoa in semen pellet of NOA patients. While the previously suggested soluble markers for spermatozoa retrieval suffered from low diagnostic specificity, our multi-step gating strategy and visualization of AKAP4+/ACRV1+/Hoechst+ cells bearing elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. Pending further validation, our assay may emerge as a non-invasive test to predict the retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of the severe forms of male infertility.

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“…Analytical proteomics and the high-quality assay to measure low-abundance proteins in blood serum and proximal fluids are indispensable tools to verify and validate novel biomarkers, including presumably highly prostate tissue-specific genes such RLN1. The knowledge of the reference values and ranges of proteins in healthy population is imperative to enable identification and validation of novel disease biomarkers [84][85][86] .…”

Section: Due To Immunoassay Cross-reactivity and Uncertainty About Ex...mentioning

confidence: 99%

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

Rais,

Drabovich

2024

Preprint

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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 the 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 the investigation of the physiological and pathological roles of REL1 and REL2 peptide hormones.

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Protein Biomarker Discovery

Cited by 9 publications

References 122 publications

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

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

Germ cell-specific proteins ACRV1 and AKAP4 facilitate identification of rare spermatozoa in semen of non-obstructive azoospermia patients

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

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