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.

Redefining serological diagnostics with immunoaffinity proteomics.

Serological diagnostics is generally defined as the detection of specific human immunoglobulins developed against viral, bacterial, or parasitic diseases. Serological tests facilitate the detection of past infections, evaluate immune status, and provide prognostic information. Serological assays were traditionally implemented as indirect immunoassays, and their design has not changed for decades.

Germ Cell-Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific 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, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation.

Assessment of risks, implications, and opportunities of waterborne neurotoxic pesticides.

Pesticides are a well-known family of chemicals that have contaminated water systems globally. Four common subfamilies of pesticides include organochlorines, organophosphates, pyrethroids, and carbamate insecticides which have been shown to adversely affect the human nervous system. Studies have shown a link between pesticide exposure and decreased viability, proliferation, migration, and differentiation of murine neural stem cells.

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.

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.

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.

Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients.

Seminal plasma, because of its proximity to prostate, is a promising fluid for biomarker discovery and noninvasive diagnostics. In this study, we investigated if seminal plasma proteins could increase diagnostic specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers. To select 147 most promising biomarker candidates, we combined proteins identified through five independent experimental or data mining approaches: tissue transcriptomics, seminal plasma proteomics, cell line secretomics, tissue specificity, and androgen regulation.

Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974.

TEX101 is a germ-cell-specific protein 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 due to the lack of human germ cell lines.

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.

Targeted Mass Spectrometry-Based Assays for Relative Quantification of 30 Brain-Related Proteins and Their Clinical Applications.

Cerebrospinal fluid (CSF) is a promising clinical sample for identification of novel biomarkers for various neurological disorders. Considering its direct contact with brain tissue, CSF represents a valuable source of brain-related and brain-specific proteins. Multiple sclerosis is an inflammatory, demyelinating neurological disease affecting the central nervous system, and so far there are no diagnostic or prognostic disease specific biomarkers available in the clinic.

Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility.

Background: TEX101 is a cell membrane protein exclusively expressed by testicular germ cells and shed into seminal plasma. We previously verified human TEX101 as a biomarker for the differential diagnosis of azoospermia, and developed a first-of-its-kind TEX101 ELISA. To demonstrate the clinical utility of TEX101, in this work we aimed at evaluating ELISA performance in a large population of fertile, subfertile, and infertile men.

Quantification of angiotensin II-regulated proteins in urine of patients with polycystic and other chronic kidney diseases by selected reaction monitoring.

Background: Angiotensin-II (Ang II) mediates progression of autosomal-dominant polycystic kidney disease (ADPKD) and other chronic kidney diseases (CKD). However, markers of kidney Ang II activity are lacking. We previously defined 83 Ang II-regulated proteins in vitro, which reflected kidney Ang II activity in vivo.

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA).

Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas.

Background: Cerebrospinal fluid (CSF) is a proximal fluid which communicates closely with brain tissue, contains numerous brain-derived proteins and thus represents a promising fluid for discovery of biomarkers of central nervous system (CNS) diseases. The main purpose of this study was to generate an extensive CSF proteome and define brain-related proteins identified in CSF, suitable for development of diagnostic assays.

Methods: Six non-pathological CSF samples from three female and three male individuals were selected for CSF analysis.

Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

Estrogen receptor alpha (ERα)-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce a secondary response to stimulation. To differentiate between the primary and secondary target proteins of ERα signaling, we measured dynamics of protein expression induced by 17β-estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by stable isotope labeling by amino acids in cell culture (SILAC) resulted in identification of 103 estrogen-regulated proteins, with only 40 of the corresponding genes having estrogen response elements.

Seminal biomarkers for the evaluation of male infertility.

For men struggling to conceive with their partners, diagnostic tools are limited and often consist of only a standard semen analysis. This baseline test serves as a crude estimation of male fertility, leaving patients and clinicians in need of additional diagnostic biomarkers. Seminal fluid contains the highest concentration of molecules from the male reproductive glands, therefore, this review focuses on current and novel seminal biomarkers in certain male infertility scenarios, including natural fertility, differentiating azoospermia etiologies, and predicting assisted reproductive technique success.

Immunocapture-Selected Reaction Monitoring Screening Facilitates the Development of ELISA for the Measurement of Native TEX101 in Biological Fluids.

Monoclonal antibodies that bind the native conformation of proteins are indispensable reagents for the development of immunoassays, production of therapeutic antibodies and delineating protein interaction networks by affinity purification-mass spectrometry. Antibodies generated against short peptides, protein fragments, or even full length recombinant proteins may not bind the native protein form in biological fluids, thus limiting their utility. Here, we report the application of immunocapture coupled with selected reaction monitoring measurements (immunocapture-SRM), in the rapid screening of hybridoma culture supernatants for monoclonal antibodies that bind the native protein conformation.

Toward an integrated pipeline for protein biomarker development.

Protein biomarker development is a multidisciplinary task involving basic, translational and clinical research. Integration of multidisciplinary efforts in a single pipeline is challenging, but crucial to facilitate rational discovery of protein biomarkers and alleviate existing disappointments in the field. In this review, we discuss in detail individual phases of biomarker development pipeline, such as biomarker candidate identification, verification and validation.

Seminal plasma as a diagnostic fluid for male reproductive system disorders.

Molecular biomarkers hold promise to advance the noninvasive diagnosis of male reproductive system disorders and facilitate the identification and management of these conditions through screening, early diagnosis and more accurate prognosis. Seminal plasma has great potential as a proximal fluid for protein biomarker discovery and as a clinical sample for noninvasive diagnostics. The seminal plasma proteome contains thousands of proteins and includes a large number of tissue-specific proteins that might accurately indicate a pathological process in the tissue of origin.

Mechanisms of Androgen-Independent Prostate Cancer.

Prostate cancer is the second leading cause of cancer-related deaths among men in North America. Almost all prostate cancers begin in an androgen-dependent state, so androgen deprivation therapy is administered and results in improved clinical outcomes. However, over time, some cancerous cells are able to survive and grow during this treatment, resulting in androgen-independent prostate cancer.

An Emerging Role of TEX101 Protein as a Male Infertility Biomarker.

Infertility is an important aspect of human reproduction. It affects up to 15% of couples, with the male factor contributing to approximately 50% of all cases. Azoospermia is one of the most severe forms of male infertility, which is characterized by the absence of sperm in semen.

Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease.

Differential diagnosis of azoospermia with proteomic biomarkers ECM1 and TEX101 quantified in seminal plasma.

Male fertility problems range from diminished production of sperm, or oligozoospermia, to nonmeasurable levels of sperm in semen, or azoospermia, which is diagnosed in nearly 2% of men in the general population. Testicular biopsy is the only definitive diagnostic method to distinguish between obstructive (OA) and nonobstructive (NOA) azoospermia and to identify the NOA subtypes of hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome. We measured by selected reaction monitoring assay 18 biomarker candidates in 119 seminal plasma samples from men with normal spermatogenesis and azoospermia, and identified two proteins, epididymis-expressed ECM1 and testis-expressed TEX101, which differentiated OA and NOA with high specificities and sensitivities.