Federica Vincenzoni

Proteomic approach in the identification of fertility pattern in seminal plasma of fertile men

OBJECTIVE To identify a panel of common seminal proteins in human seminal plasma by fertile men that might be involved in successful reproduction. DESIGN Experimental study. SETTING University hospital. PATIENT(S) Five fertile men who conceived within 3 months before the start of the study. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Proteomic analysis performed by an Ultimate 3000 Nano/Micro-HPLC apparatus equipped with an FLM-3000-Flow manager module and coupled with an LTQ Orbitrap XL hybrid mass spectrometer; gene ontology analysis. RESULT(S) From 919 to 1,487 unique proteins were identified per individual subject sample. Among these proteins, 83 proteins were present in all samples, including some proteins that might be involved in male fertility, such as semenogelin I, semenogelin II, olfactory receptor 5R1, lactoferrin, hCAP18, spindling, and clusterin. The gene ontology annotation analysis provided further information in describing common pattern in male fertility. CONCLUSION(S) The identification of common seminal plasma proteome in fertile men could provide better insight into the physiology of male fertility and might identify novel markers of male infertility.

Globular structure of human ovulatory cervical mucus.

Human cervical mucus is a heterogeneous mixture of mucin glycoproteins whose relative concentration changes during the ovulatory phases, thereby producing different mucus aggregation structures that can periodically permit the transit of spermatozoa for fertilization. In preovulatory phase, mucus is arranged in compact fiber‐like structures where sperm transit is hindered. Previously, through observations made of fixed and dehydrated samples, a permissive structure in the ovulatory phase was attributed to the larger diameters of pores in the mucus network. Instead, by means of atomic force microscopy, we can show, for the first time, that unfixed ovulatory mucus is composed by floating globules of mucin aggregates. This finding sheds new light on the mechanism that governs spermatozoa transit toward the uterine cavity. In addition, we demonstrate that the switch from globular ovulatory to fibrous preovulatory mucus largely depends on a pH‐driven mechanism. Analysis of mucin 5B primary sequence, the main mucin in ovulatory mucus, highlights pH‐sensitive domains that are associated to flexible regions prone to drive aggregation. We suggest an involvement of these domains in the fiber‐to‐globule switch in cervical mucus.— Brunelli R., Papi, M., Arcovito, G., Bompiani, A., Castagnola, M., Parasassi, T., Sampaolese, B., Vincenzoni, F., De Spirito M. Globular structure of human ovulatory cervical mucus. FASEB J. 21, 3872–3876 (2007)

Characterization of dendrimer properties by capillary electrophoresis and their use as pseudostationary phases

The general properties of dendrimers and in particular their electrolytic characteristics that are relevant in electrokinetic separations, are described. In order to confirm theoretical considerations on commercial dendrimer charge and hydrodynamic radius, several capillary zone electrophoresis (CZE) experiments were performed. Electrophoretic mobilities measured at different pH values indicated a sensible increase of dendrimer hydrodynamic radius at pH values lower than 2.5. This was probably due to the Coulombic repulsion of charged amine groups of the inner dendrimer shells. The principal reasons that should address the use of dendrimers as pseudostationary phases in micellar electrokinetic chromatography (MEKC) are discussed. Moreover, a survey of different separations performed utilizing dendrimers in MEKC as well as of several future plausible uses of various classes of dendrimers is presented.

Unraveling the different proteomic platforms

This review is addressed to scientists working outside the field of proteomics and wishes to shed a light on the possibility offered by the latest proteomics strategies. Bottom-up and top-down platforms are critically examined outlining advantages and limitations of their application to qualitative and quantitative investigations. Discovery, directed and targeted proteomics as different options for the management of the MS instrument are defined emphasizing their integration in the experimental plan to accomplish meaningful results. The issue of data validation is analyzed and discussed. The most common qualitative proteomic platforms are described, with a particular emphasis on enrichment methods to elucidate PTMs codes (i.e. ubiquitin and histone codes). Label-free and labeled methods for relative and absolute quantification are critically compared. The possible contribution of proteomics platforms to the transition from structural proteomics to functional proteomics (study of the functional connections between different proteins) and to the challenging system biology (integrated study of all the functional cellular functions) is also briefly discussed.

Proteomics of human seminal plasma: Identification of biomarker candidates for fertility and infertility and the evolution of technology

Proteomics is a research area that has developed rapidly in the last decade. It studies the large‐scale characterization of the full protein components of a cell, a tissue, or a biological fluid. In the last decade, clinical proteomics has developed new technology and bioinformatics useful in identifying molecular markers of pathology; the next decade might be the era of proteomics. Seminal plasma (SP) represents a good sample for proteomic analysis in the evaluation of male fertility/infertility. SP is an acellular fluid conglomerate, comprised of contributions from the epididymis and accessory sexual glands. Human SP contains many proteins that are important to the successful fertilization of the oocyte by the spermatozoa. Proteomic studies have identified numerous seminal‐specific proteins, and recent reports have provided a further understanding of their function with respect to male fertility. Upon further validation, these proteins may be useful in the clinical distinction between fertility and infertility. This article reviews the proteomic methods, such as one dimensional polyacrylamide gel electrophoresis (1D–PAGE), two‐dimensional polyacrylamide gel electrophoresis (2D–PAGE), and mass spectrometry (MS), employed to detect human SP markers involved in fertility and infertility. As such, proteomic studies will help the development of new techniques to identify novel biomarkers for a better clinical diagnosis and treatment of male infertility. Mol. Reprod. Dev. 80: 350–357, 2013.

Top-down platform for deciphering the human salivary proteome.

Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids.

O2-dependent stimulation of the pentose phosphate pathway by S-nitrosocysteine in human erythrocytes ☆

In the present study we analysed the effects of S-nitrosocysteine (CysNO) on adult human red blood cell metabolism and observed that metabolic response depended on the degree of cell oxygenation. In particular, glucose metabolised through the pentose phosphate pathway (PPP) was higher in treated erythrocytes than in untreated cells only at high O(2) pressure. Since, following the treatment of intact cells with CysNO, glucose-6-phosphate dehydrogenase (G6PD) and phosphofructokinase (PFK) activities did not evidence any significant alteration, the possibility that the stimulation of PPP was triggered by a CysNO mediated modification of these enzymes was excluded. Intracellular S-nitrosoglutathione (GSNO), detected only in treated red blood cells, may be linked solely to the exposition to the NO donor. A possible rationalisation of the different metabolic behaviour shown by erythrocytes as a function of their oxygenation state is proposed. It takes into account the different route of catabolic degradation observed in vitro for GSNO under aerobic and anaerobic condition.

Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis

A method for separation and quantification of S‐nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25°C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S‐nitrosoglutathione analyses were checked. The results evidenced that S‐nitrosoglutathione is formed in erythrocytes treated with S‐nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.

Proteomic characterization of pediatric craniopharyngioma intracystic fluid by LC‐MS top‐down/bottom‐up integrated approaches

The combination of top‐down and bottom‐up platforms was utilized for the LC‐MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high‐resolution LC‐ESI‐LTQ‐Orbitrap‐MS analysis while low‐resolution LC‐ESI‐IT‐MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top‐down analyses were applied to liquid/liquid extracted samples while bottom‐up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A‐I, A‐II, C‐I and J, hemoglobin fragments, ubiquitin, α‐2‐HS‐glycoprotein or fetuin A, α‐1‐antichymotrypsin, vitamin D binding protein, and α‐1‐acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments.

Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to β- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes.