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A suboptimal blood vitamin C (ascorbate) level increases the risk of several chronic diseases. However, the detection of hypovitaminosis C is not a simple task, as ascorbate is unstable in blood samples. In this study, we examined the serum proteome of mice lacking the gulonolactone oxidase (Gulo) required for the ascorbate biosynthesis. Gulo-/- mice were supplemented with different concentrations of ascorbate in drinking water, and serum was collected to identify proteins correlating with serum ascorbate levels using an unbiased label-free liquid chromatography-tandem mass spectrometry global quantitative proteomic approach. Parallel reaction monitoring was performed to validate the correlations. We uncovered that the serum proteome profiles differ significantly between male and female mice. Also, unlike Gulo-/- males, a four-week ascorbate treatment did not entirely re-establish the serum proteome profile of ascorbate-deficient Gulo-/- females to the optimal profile exhibited by Gulo-/- females that never experienced an ascorbate deficiency. Finally, the serum proteins involved in retinoid metabolism, cholesterol, and lipid transport were similarly affected by ascorbate levels in males and females. In contrast, the proteins regulating serum peptidases and the protein of the acute phase response were different between males and females. These proteins are potential biomarkers correlating with blood ascorbate levels and require further study in standard clinical settings. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD027019.

Mammalian semen contains a heterogeneous population of sperm cells. This heterogeneity results from variability in the complex processes of cell differentiation in the testis, biochemical modifications undergone by spermatozoa during transit along the male reproductive tract, interactions with secretions from accessory sex glands at ejaculation, and, in the context of reproductive technologies, in the ability of ejaculated spermatozoa to resist damage associated with freeze-thaw procedures. When submitted to density gradient centrifugation, ejaculated spermatozoa distribute themselves into two distinct populations: a low-density population characterized by low motility parameters, and a high-density population with high motility characteristics. To understand the origin of ejaculated spermatozoa heterogeneity, cryopreserved semen samples from bulls used by the artificial insemination (A.I.) industry were submitted to Percoll gradient centrifugation. Proteins from low and high density spermatozoa were then extracted with sodium deoxycholate and submitted to proteomic analysis using iTRAQ (isobaric tag for relative and absolute quantitation) methodologies. Quantification of selected sperm proteins was confirmed by multiple reaction monitoring (MRM). Overall, 31 different proteins were more abundant in low-density spermatozoa, while 80 different proteins were more abundant in the high-density subpopulation. Proteins enriched in high-density spermatozoa were markers of sperm functionality such as the glycolytic process, binding to the egg zona pellucida, and motility. Low-density spermatozoa were not solely characterized by loss of proteins and their associated functions. Chaperonin-containing TCP1s and chaperones are hallmarks of the low-density subpopulation. iTRAQ analysis revealed that other proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1, and clusterin are overexpressed in low-density spermatozoa suggesting that these proteins represent defects occurring at different steps during the sperm journey. These differences contribute to the sperm cell heterogeneity present in mammalian semen.

Male factors account for 40% of infertility cases. The identification of differentially expressed proteins on spermatozoa from fertile and infertile men can help in the elucidation of the molecular basis of male infertility. The aim of this study was to compare sperm proteomes from 3 different groups: fertile men, normozoospermic men consulting for infertility, and normozoospermic men with an impaired capacity for fertilization (IVF-failure). We used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and LC-MS analysis to identify proteins that are differentially expressed. A total of 348 unique proteins were identified and quantified. The analysis identified 33 proteins that were differentially expressed in the IVF-failure group vs the fertile group. Comparison of the infertile and fertile groups revealed that 18 proteins appeared to be differentially expressed. Four proteins were similarly altered in the IVF-failure and infertile groups: semenogelin 1 (SEMG1), prolactin-induced protein (PIP), glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), and phosphoglycerate kinase 2 (PGK2). These protein markers were selected for validation using multiple reactions monitoring mass spectrometry (MRM-MS) and further confirmed by Western blot analysis. Overall, these results suggest that a panel of proteins may be used as biomarkers for future studies of infertility.

The prostate is a relatively homogeneous tissue that is highly specialized in synthetic and secretory functions. The frequency of malignant growth explains its great clinical significance. We used here a combination of subcellular fractionation, 1-DE (one-dimensional gel electrophoresis) protein separation and mass spectrometry, to establish a prostate protein expression profile in mice. Analysis of proteins present in cytosolic (C) and membrane (P) prostate fractions led to the identification of 619 distinct proteins. A majority of abundant proteins were found to compose the metabolism and protein synthesis machinery. Those identified also correspond to known endoplasmic reticulum and Golgi residents, chaperones and anterograde cargos. They included a series of proteins involved in exocytic/endocytic trafficking. Among the signaling proteins, we identified the ubiquitin-like peptides smt3. We showed that both free small ubiquitin-related modifier SUMO-2/3 and SUMO-1 levels are subject to tight control by the androgen 5alpha-dihydrotestosterone (DHT). By contrast with SUMO-2/3, free SUMO-1 peptides are particularly abundant in the prostate when compared with other tissues. Therefore, we report prostate protein expression profiles of cytosolic and membrane fractions in mice. Our data suggest that the identified free SUMO peptides play an important role in this secretory tissue.