Objectives: Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). Design: Proteins from OA cartilage taken at knee arthroplasty (n = 6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved Ntermini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/ blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. Results: We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. Conclusions: OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers. (c) 2022 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The seminal plasma protein profile is a reflection of the spermatozoa homeostasis. This study aimed to validate, using Western Blot (WB), potential biomarkers identified in the seminal plasma of men with primary or secondary infertility in comparison with fertile donors. This study includes 18 sperm samples from proven fertile men, men with primary infertility and men with secondary infertility (n=6/group). Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the differently expressed proteins (DEPs) in the infertile group and key DEPs were validated using WB. Proteomic profiles of seminal plasma from fertile men, men with primary or secondary infertility were obtained by mass-spectrometry. Based on the functional bioinformatics analysis using IPA, Metacore and public annotation databases key DEPs associated with infertility related processes were selected for validation by WB. For primary infertility, the chosen proteins were annexin A2, transferrin, cell division control protein 42, CD63, peroxiredoxin 2, semenogelin 1 and 2. For secondary infertility, we selected annexin A2, complement C4, complement C3, heat shock protein 70, semenogelin 1 and amyloid precursor protein. The statistical analysis of WB results was performed in the MedCalc using a Mann-Whitney test (p<0.05). Our validation using WB showed an overexpression of annexin A2 (p=0.03) in men with primary infertility when compared to fertile donors, which was concordant with the mass spectrometry results. In men with secondary infertility, we observed an underexpression of complement C3 (p=0.03) relative to fertile men. However, this result did not match with the proteomic analysis that indicated an overexpression of complement C3. Annexin A2, which is involved in sperm binding, may be a potential biomarker for the diagnosis of men with primary infertility. Further validation of additional proteins is necessary to identify other candidate proteins that may also be implicated in male infertility.

Alteration in expression levels of seminal plasma proteins may affect the fertilizing ability of spermatozoa. This study aimed to 1) compare seminal plasma proteome of proven fertile men with infertile varicocele patients; and 2) identify protein biomarkers in seminal plasma of infertile men with unilateral and bilateral varicocele. Proteomic profiling of seminal plasma was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Key differentially expressed proteins (DEPs) in varicocele (unilateral and bilateral) patients and fertile men were selected using bioinformatics analysis and evaluated using western blot (WB). Pooled seminal plasma samples from fertile men (n=5), unilateral (n=5), and bilateral (n=5) varicocele patients were processed for quantitative proteomic analysis. Proteins identified by LC-MS/MS in both varicocele groups were combined and compared with the fertile group. Ingenuity pathway analysis software was used to narrow down key DEPs associated with normal sperm function. Four proteins namely acrosin (ACR), heat shock-related 70 kDa protein 2 (HSPA2), peroxiredoxin 2 (PRDX2) and apolipoprotein A2 (APOA2) were validated by WB and evaluated in fertile men (n=6) and varicocele patients (n=12). Statistical significance was calculated using Mann-Whitney test. A total of 412 and 486 proteins were detected in seminal plasma of fertile men and varicocele patients respectively. Twenty eight proteins were identified as DEPs and key DEPs selected for WB validation were associated with binding of zona pellucida (ACR), folding of proteins (HSPA2), oxidative stress (PRDX2), lipid peroxidation and DNA fragmentation (APOA2). Expression and fold change of these proteins are shown in Table 1. Irrespective of varicocele type (unilateral or bilateral), seminal plasma protein profiles of infertile varicocele patients differ from that of fertile healthy men. We propose proteins APOA2, HSPA2 and PRDX2 as potential biomarkers for evaluating the fertility status of varicocele patients.Tabled 1Table 1Validation of DEPs in varicocele patients by western blotProteinExpressionRelative Fold ChangeP-valueACR↓0.810.3861HSPA2↓0.100.0037*PRDX2↑1.290.0474*APOA2↓0.670.0373**Statistically significant (P<0.05), ↓ underexpressed, ↑ overexpressed Open table in a new tab

POTENTIAL BIOMARKERS FOR MEN WITH PRIMARY INFERTILITY Ana Dias Martins1,2, Ashok Agarwal1, Luna Samanta3, Rakesh Sharma1, Banu Gopalan4, Belinda Willard5, Zhihong Cui6, Damayanthi Durairajanayagam7, Edmund Sabanegh8 1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, U.S.A. 2Dep. Microscopia Lab. de Biologia Celular and Unit for Multidisciplinary Research in Biomedicine, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal. 3Department of Zoology, Ravenshaw University, Odisha, India. 4Bioinformatics, Yorg Corporation, Plano, U.S.A. 5Research Core Services Proteomics, Cleveland Clinic, Cleveland, U.S.A. 6College of Pharmaceutical Sciences, Southwest University, Chongqing, China. 7Discipline of Physiology-Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia. 8Urology, Cleveland Clinic, Cleveland, U.S.A.

PROTEINS IN SEMINAL PLASMA OF INFERTILE MEN WITH UNILATERAL VARICOCELE Manesh Kumar Panner Selvam1, Ashok Agarwal1, Luna Samanta2, Rakesh Sharma1, Sajal Gupta1, Damayanthi Durairajanayagam3, Zhihong Cui4, Ahmet Ayaz5, Banu Gopalan6, Belinda Willard7, Edmund Sabanegh8 1American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, U.S.A. 2Department of Zoology, Ravenshaw University, Odisha, India. 3Discipline of Physiology-Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia. 4College of Pharmaceutical Sciences, Southwest University, Chongqing, China. 5Department of Urology, Tulane Medical Center, New Orleans, U.S.A. 6Bioinformatics, Yorg Corporation, Plano, U.S.A. 7Research Core Services Proteomics, Cleveland Clinic, Cleveland, U.S.A. 8Urology, Cleveland Clinic, Cleveland, U.S.A.

The Hodgkin's lymphoma (HD) is one of the most common cancers affecting men in reproductive age. This study aims to analyze sperm parameters and alterations in proteomic profiles of spermatozoa from men with HD undergoing sperm banking before cancer therapy. Evaluation of sperm parameters and proteomic profiles of men with HD. We analyzed the sperm parameters in fertile men (donor) (n=42) and men with HD before cancer therapy (patients) (n=38). We used Mann-Whitney test to deduce statistical differences in sperm parameters (p<0.05). We compared the proteomic profiles of spermatozoa from donors (n=3) and patients (n=3) using LTQ-Orbitrap Elite hybrid MS system. Samples were analyzed using Mascot and SEQUEST software to search the Human Reference Sequence database. The identified differently expressed proteins (DEPs) were evaluated using Ingenuity Pathway Analysis (IPA). We observed a lower concentration, total count, and total motile spermatozoa in patients compared to donors. Global proteomic analysis identify a total of 1169 proteins. Among the 134 identified DEPs: 35 were overexpressed: 80 were underexpressed; 16 were unique to donors; and 3 were unique to patients. The IPA analysis revealed that proteins involved in capacitation, acrosome reaction, binding of sperm to the zona pellucida, sperm motility, regulation of sperm DNA damage, and apoptosis were significantly downregulated in HD. Cancer and reproductive system disease were the top significantly regulated diseases and disorders in the spermatozoa of patients. The molecular and cellular functions such as cell-to-cell signaling and interaction, cellular assembly and organization, and protein modifications were significantly impaired in the spermatozoa of patients. Heat shock factor proteins 1 and 2; and 1,2-dithiol-3-thione were predicted as the top inhibited upstream regulators. Semen quality was significantly decreased in men with HD relative to fertile men. However, proteomic data showed an altered proteome in spermatozoa of men with HD, which may explain how these alterations can potentially compromise the fertility in these men and help identify therapeutic strategies to increase the quality of sperm and successful pregnancies.

The quantitative assessment of the synthesis of individual proteins has been greatly hin‐ dered by the lack of a high-throughput nonradioactive method. We recently developed a method that we call “proteome dynamics” and software that enables high-throughput ki‐ netic analyses of peptides on a proteome-wide scale. Previous studies established that or‐ al administration of heavy water ( 2 H 2 O or deuterium oxide, D 2 O) is safe and well tolerated in humans. Briefly, a loading dose of 2 H 2 O, a nonradioactive isotope, is adminis‐ tered in drinking water. 2 H 2 O rapidly labels body water and transfers 2 H from 2 H 2 O to 2 H-labeled amino acids, which incorporates into proteins dependent upon the rate of syn‐ thesis of the specific protein. Proteins are analyzed by high-resolution mass spectrometry and protein synthesis is calculated using specialized software. We have established the effectiveness of this method for plasma and mitochondrial proteins. We demonstrated that fasting has a differential effect on the synthesis rates of proteins. We also applied this method to assess the effect of heart failure on the stability of mitochondrial proteins. In this review, we describe the study design, instrumentation, data analysis, and biological application of heavy water-based proteome turnover studies. We summarize this chapter with the challenges in the field and future directions. The effects of dietary factors on tissue protein synthesis were investigated in acute fasting (20 h) vs. chronic food restriction (7 days), and feeding (a single meal) conditions in rats. Both acute and chronic fasting significantly reduced mixed tissue protein synthesis in the liver and gastrocnemius muscle, while it did not affect protein synthesis in the left ventricle of the heart [32], indicating that cardiac protein synthesis is preserved in conditions of nutritional pertur‐ bations. The follow-up studies demonstrated that diet-induced obesity in mice did not affect the skeletal muscle protein synthesis; however, it did impair the response of muscle protein synthesis to nutrient supply [34].

Varicocele the most common correctable cause of male factor infertility is observed in 35% to 50% of adult men with primary infertility. Oxidative stress plays an important role in the pathogenesis of sperm DNA damage in patients with varicocele. The objective was to examine if proteomic analysis and bioinformatic tools can help in identifying proteins of interest in infertile men with unilateral varicocele. This prospective study analyzed spermatozoa proteins from infertile men with unilateral varicocele (n = 5) and healthy fertile men (n = 5) who recently established a pregnancy to identify the proteins of interest involved in the pathophysiology of varicocele and male infertility. Spermatozoa were obtained from infertile men with unilateral varicocele and healthy men of proven fertility. Proteins were extracted and separated by 1-D gel. Bands were digested and on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. A total of 1035 proteins were identified in the spermatozoa of fertile group and 757 in the unilateral varicocele group. 369 proteins were differentially expressed. Of these, 120 were unique to the fertile group and 38 to the unilateral varicocele group. 114 were overexpressed and 97 were underexpressed in the unilateral varicocele group. Apoptosis, post-translational modification, protein ubiquitination, mitochondrial dysfunction, oxidative stress response were some of the major functional categories observed for the differentially expressed proteins. Additionally, we have identified about 30 proteins of interest that play a role in sperm motility, capacitation, acrosome reaction and fertilization and may be compromised or altered in infertile men with unilateral varicocele. We identified significant differences in the distribution of differentially expressed proteins (DEP) in fertile men and infertile men with unilateral varicocele. Distinct proteins are involved in various biological processes, molecular function and cellular location. These proteins may serve as potential biomarkers that are involved in the pathophysiology of varicocele and male infertility.

Varicoceles appear to affect later stages of spermatogenesis and cause scrotal hyperthermia, hypoxia, hormonal imbalances, and re-flow of metabolites from renal and/or adrenal glands leading to oxidative stress. The objective was to study the major differences in the distribution of spermatozoa proteins in infertile men with varicocele compared to fertile men. his prospective proteomic study analyzed proteins in spermatozoa from infertile men with unilateral (n=5) or bilateral (n=3) varicocele and men of proven fertility (n=5) to study the proteins involved in the pathophysiology of varicocele-associated male infertility. Spermatozoa proteins were extracted from infertile men with unilateral and bilateral varicoceles and men with proven fertility. Proteins were separated by 1-D gel electrophoresis and bands were digested and identified on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Bioinformatics tools were used to identify pathways and functions of proteins of interest. Of the 99 proteins that were differentially expressed in the varicocele group, 9 proteins were uniquely expressed in the fertile group compared to 2 proteins that were unique to the varicocele group. 12 proteins were overexpressed and 76 were underexpressed in the varicocele group. In the varicocele group, the top networks were energy production, lipid metabolism, post-translational modification and protein folding. While proteins such as outer dense fiber protein 2 isoform 3 and tektin-3, that are known to play a role in sperm motility were overexpressed in the varicocele group, a majority of proteins, such as, acrosin binding protein precursor, calmegin precursor that are involved in spermatid differentiation, spermatid development, spermatogenesis, reproductive cellular processes, and fertility were observed to be underexpressed. Proteomics and bioinformatic analysis are powerful tools in understanding the pathology of varicocele associated male infertility. We have identified key proteins that are altered or modified in the presence of varicocele and may result in male infertility. Infertile men with unilateral or bilateral varicocele have a large number of spermatozoal proteins that are differentially expressed compared to those of fertile men. These protein alterations may be a contributing factor in male infertility.

γ-Carboxylated Glu (Gla) is a post-translational modification required for the activity of vitamin K-dependent (VKD) proteins that has been difficult to study by mass spectrometry due to the properties of this negatively charged residue. Gla is generated by a single enzyme, the γ-glutamyl carboxylase, which has broad biological impact because VKD proteins have diverse functions that include hemostasis, apoptosis, and growth control. The carboxylase also contains Glas, of unknown function, and is an integral membrane protein with poor sequence coverage. To locate these Glas, we first established methods that resulted in high coverage (92%) of uncarboxylated carboxylase. Subsequent analysis of carboxylated carboxylase identified a Gla peptide (729-758) and a missing region (625-647) that was detected in uncarboxylated carboxylase. We therefore developed an approach to methylate Gla, which efficiently neutralized Gla and improved mass spectrometric analysis. Methylation eliminated CO2 loss from Gla, increased the ionization of Gla-containing peptide, and appeared to facilitate trypsin digestion. Methylation of a carboxylated carboxylase tryptic digest identified Glas in the 625-647 peptide. These studies provide valuable information for testing the function of carboxylase carboxylation. The methylation approach for studying Gla by mass spectrometry is an important advance that will be broadly applicable to analyzing other VKD proteins.

Alpha-2-macroglobulin (a2M), a 720-kDa plasma protein, functions as an extracellular proteinase inhibitor and carrier of cytokines, growth factors and hormones. Disulfide bonds are susceptible for thiol-exchange reactions with homocysteine (Hcy). The structure of a2M contains 96 cysteine residues and one can assume that some of these disulfide bridges might interact with Hcy. Using 35S-D,L-homocysteine, we show that a2M is homocysteinylated in both its native and in the protein-proteinase complex (“activated” a2M). The stoichiometry of Hcy binding to native and activated a2M is dose-dependent: treatment of native and activated a2M with 25 and 50 μM 35S-D,L-Hcy at 37°C for 4 h yielded approximate Hcy:a2M ratios of 2:1 and 4:1 for native a2M, and 3:1 and 6:1 for activated a2M. Trypsin-protein esterase activity of a2M was shown to be decreased by 12–35% in homocysteinylated-a2M compared to untreated a2M, suggesting that high levels of Hcy in the blood may affect the proteinase-binding function of a2M. This might explain why proteolytic activity remains high in inflammatory tissue fluids and why cytokines and growth factors that usually bind to a2M continue to play important roles in the progression of inflammation and atherosclerosis.

Senescence represents the last stage of flower development, ultimately culminating in the death of the petals. The senescence program is regulated by coordinated changes in gene and protein expression, and the later stages of senescence share many characteristics of programmed cell death.