Ion mobility mass spectrometry: an elegant alternative focusing on speciation studies

This work is proposed to demonstrate the Traveling-Wave Ion Mobility Specrometry (TWIMS) coupled to Mass Spectrometry (MS) as an alternative technique for speciation analysis between metals/metalloids and biomolecules. Mobilities of bovine carbonic anhydrase bound to Ba(2+), Cu(2+), Pb(2+), Zn(2+), Cr(3+), Cr(6+), Se(4+) and Se(6+) were estimated. The metal belonging to the bovine carbonic anhydrase structure, commonly found in the commercially available enzyme, was removed by filtration, using centrifugal filter devices. Then, some metals/metalloids were added to 10.0 mmol L(-1) ammonium acetate at pH = 6.8 enzyme solution. Experiments were carried out by direct insertion of the sample at 10 mu L min(-1) flow rate into the ESI source of the instrument. Carbonic anhydrase mobility varied according to the metal bound in its structure, following the order: Zn(2+) < Cu(2+) < Ba(2+) < Pb(2+). Metals with higher affinity by the enzyme, such as Zn(2+) and Cu(2+) had lower mobility, suggesting a higher structural modification, binding itself to the enzyme metallic site. Considering metals with different oxidation states, the enzyme mobility followed the order: Se(4+) < Cr(6+) < Se(6+) < Cr(3+).26120120

A new label-free approach for the determination of reaction rates in oxidative footprinting experiments

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Hydroxyl radical-mediated oxidative footprinting coupled to mass spectrometric analysis is an attractive technique for protein surface mapping, conformational changes monitoring, and protein-ligand interfaces mapping in solution. In this technique, a protein is oxidized by in situ-generated hydroxy radicals and the site and rate of oxidation can be determined by proteolysis followed by mass spectrometric analysis. Changes in peptide oxidation rate can then be correlated to the changes in solvent exposure, and information about conformational changes or interaction domains can be obtained. The method relies, therefore, on the accurate measurements of peptide oxidation rate. Here, we describe a new label-free method to determine the oxidation rate of peptides that is based on the consumption of the unoxidized peptide instead of measuring the formation of oxidized peptides. The reaction rate thus obtained presents a better linearity and lower variation when compared to the traditional method. The label-free method is also simpler to implement and automation can be achieved through label-free quantitation software.4052476797686Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Sao Paulo Proteomics Network (FINEP) [01.07.0290.00]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2004/14846-0]Sao Paulo Proteomics Network (FINEP) [01.07.0290.00]FAPESP [08/57805-2]CNPq [573672/2008-3

Ion mobility spectrometry focusing on speciation analysis of metals/metalloids bound to carbonic anhydrase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)In the present work, traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) was applied to speciation analysis of metalloproteins. The influence of pH on complexation conditions between some metals and bovine carbonic anhydrase was evaluated from pH 6 to 9, as well as the time involved in their complexation (0-24 h). Employing TWIMS-MS, two conformational states of bovine carbonic anhydrase were observed with charge states of +12 and +11; these configurations being evaluated in terms of the folded state of the apo form and this protein (at charge state +11) being linked to barium, lead, copper, and zinc in their divalent forms. Metalloprotein speciation analysis was carried out for copper (Cu+ and Cu2+), lead (Pb2+ and Pb4+), and selenium (Se4+ and Se6+) species complexed with bovine carbonic anhydrase. Mobilities of all complexed species were compared, also considering the apo form of this protein.4052476537660Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Traveling-Wave Ion Mobility Mass Spectrometry Analysis of Isomeric Modified Peptides Arising from Chemical Cross-Linking

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Traveling-wave ion mobility (TWIM) coupled to mass spectrometry (MS) has emerged as a powerful tool for structural and conformational analysis of proteins and peptides, allowing the analysis of isomeric peptides (or proteins) with the same sequence but modified at different residues This work demonstrates the use of the novel TWIM-MS technique to separate isomeric peptide ions derived from chemical cross-linking experiments, which enables the acquisition of distinct product ion spectra for each isomer, clearly indicating modification on different sites Experiments were performed with four synthetic peptides, for which variable degrees of mobility separation were achieved In cases of partially overlapping mobility arrival time distributions (ATDs), extracting the ATDs of fragment ions belonging to each individual isomer allowed their separation into two distinct ATDs Accumulation over regions from the specific ATDs generates the product ion spectrum of each isomer, or a spectrum highly enriched in their fragments The population of both modified peptide isomers was correlated with the intrinsic reactivities of different Lys residues from reactions conducted at different pH conditions (J Am Soc Mass Spectrom 2010, 21, 2062-2069) (C) 2010 Published by Elsevier Inc on behalf of American Society for Mass Spectrometry211220622069Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [FAPESP 2004/14,846-0, FINEP 01 07 0290 00]FAPESP [FAPESP 08/57,805-2, CNPq 573672/2008-3

Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (20062010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking. Funding for the study was provided by Fundao de Amparo Pesquisa do Estado de So Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the So Paulo Federal University.271131403149Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Division of Urology, Human Reproduction Section at the Sao Paulo Federal UniversityFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2007/59423-7

The follicular microenviroment as a predictor of pregnancy: MALDI-TOF MS lipid profile in cumulus cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)This research proposed to study the changes in lipid composition in cumulus cells (CCs) from women who achieved pregnancy compared with women who did not, after in vitro fertilization treatment. This approach has the potential to provide novel information on the lipid metabolism of the CCs and as an additional method to predict pregnancy. Fifty-four samples from couples with tubal and male factor infertility and where the female partner was age 35 or younger were divided in two groups according to their level of hCG 14 days after embryo transfer as follows: (1) 23 samples in pregnant group and (2) 31 samples in non-pregnant group. Lipid extraction was performed by the Bligh-Dyer protocol, and lipid profiles were obtained by MALDI-TOF MS. Mass spectra data were processed with MassLynx, and statistical analysis was performed using MarkerLynx extended statistic. OPLS-DA model was built. S-plot Analysis revealed three ions as potential markers in the pregnant group, and five ions in the non-pregnant group. These ions were identified in the human metabolome database (HMDB) as phosphatidylcholine in the pregnant group and as phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol species in the non-pregnant group. These lipids might be involved in cell proliferation and differentiation, apoptosis and GAP junction regulation. We conclude that MALDI-TOF MS can be used as an informative and fast analytical strategy to obtain and study the lipid profile of cumulus cells and can potentially be used as a supporting tool to predict pregnancy based on the metabolic state of the CCs.291112891297Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2010/14732-5

Lipid profiling of follicular fluid from women undergoing IVF: Young poor ovarian responders versus normal responders

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)This study identified possible lipid biomarkers in follicular fluid from women with poor ovarian response. These biomarkers indicate pathophysiological pathways and have potential diagnostic applications. An observational case-control study of young women undergoing ovarian stimulation for in-vitro fertilization was conducted. The participants were categorized into a poor ovarian response group and a normal ovarian response to stimulation group. All of the women underwent the same ovarian stimulation protocol, and follicular fluid was collected after ovarian aspiration. Analyses were performed using matrix-assisted laser desorption/ionization mass spectrometry. Principal component analysis and Volcano plots were used to describe follicular fluid classification models based on the lipid profiles. A total of 10 lipids were differentially expressed between the study and control groups. Of these lipid ions, three belonged to the phosphatidylcholine subclass and were present in higher concentrations in the control group. The other seven differential lipids were present in the study group and classified into four lipid subclasses: phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, and diacylglycerols. These distinctive lipids may be involved in hormonal responses and oocyte development processes and may be useful as biomarkers for therapeutic intervention in women with poor ovarian response.164269277Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Changes in the seminal plasma proteome of adolescents before and after varicocelectomy

Objective: To compare seminal plasma protein profiles before and after varicocele correction to assess if surgical intervention alters the protein profile. Design: Prospective study. Setting: Academic research environment. Patient(s): Nineteen adolescent boys with varicocele grades II or III. Intervention(s): Two semen samples were collected before bilateral subinguinal microsurgical varicocelectomy, and two semen samples were collected 3 months after surgery. Seminal plasma protein profiles were determined with the use of two-dimensional gel electrophoresis. Proteins were separated in 18-cm 3-10 pH strips and 10%-17.5% gradient gels. Gels were stained, scanned, and compared with the use of Imagemaster 2D platinum 7.0. Spots of interest were removed from gels, and protein digestion was performed with the use of trypsin. Digests were identified with the use of electrospray ionization-quadrupole/time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS), and spectra were analyzed with the use of the Mascot software. Main Outcome Measure(s): Proteins uniquely or overexpressed in each period (before or after varicocelectomy). Result(s): Nineteen spots were differentially expressed between pre- and postsurgery samples. Identified proteins were albumin, proteasome subunit alpha type 6, alpha-1-antitrypsin, fibronectin, CD177, prostatic acid phosphatase, specific prostatic antigen, alpha-2antiplasmin, vitamin D-binding protein, gastricsin, clusterin, semenogelin-1, semenogelin-2, superoxide dismutase, protein-glutamine gamma glutamyltransferase-4, and prolactin-inducing protein. Conclusion(s): Varicocelectomy is associated with changes in the seminal plasma protein profile. Understanding specific pathways leading to male infertility may further assist physicians in demonstrating deviation from homeostasis in male infertility. In addition, it may be possible to observe if surgical intervention does indeed revert altered pathways toward a homeostatic state. ((C) 2013 by American Society for Reproductive Medicine.)100366767

Differential seminal plasma proteome according to semen retrieval in men with spinal cord injury

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Objective: To evaluate protein expression profile and to quantify proteins present in seminal plasma from men with spinal cord injury (SCI) and healthy men without SCI. Design: Experimental study. Setting: University hospital. Patient(s): Twelve SCI patients divided into two groups, six who underwent electroejaculation (EEJ) and six who underwent penile vibratory stimulation (PVS); and ten control subjects presenting normal sperm motility and concentration. Intervention(s): EEJ and PVS. Main Outcome Measure(s): The seminal plasma protein profile was analyzed by two proteomic strategies: data-independent label-free quantitative proteomics (MSE) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). Result(s): A total of 638 different proteins were identified by MSE and 18 by 2D SDS-PAGE followed by tandem mass spectrometry. Interactome analysis showed key reproductive biologic processes-insemination, sperm and oocyte fusion, and acrosome reaction-related to all groups, as were triglyceride stimuli. Processes related to actin and muscle function and to iron oxidation, transportation, and homeostasis were found only in the EEJ and PVS groups; response to hydrogen peroxide and increased immune response was found only in the PVS group. Conclusion(s): This study was able to demonstrate differential protein expression among control, PVS, and EEJ groups; SCI is responsible for alterations in seminal plasma protein profile leading to a deviation from homeostasis; proteins reported in both PVS and EEJ groups correlate with the pathophysiology of SCI-related infertility. (Fertil Steril (R) 2013; 100: 959-69. (C) 2013 by American Society for Reproductive Medicine.)1004959+Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Proteomic analysis of follicular fluid from women with and without endometriosis: New therapeutic targets and biomarkers

Endometriosis is a gynecological disease that affects women of reproductive age. The protein profiles of women with endometriosis who were able or unable to achieve pregnancy and women without endometriosis who did achieve pregnancy were compared in this study. The follicular fluid was collected from 21 patients undergoing in vitro-fertilization treatment, according to the following groups: nine women in the control group (Group C), four women with endometriosis who achieved pregnancy (Group E.P), and eight women with endometriosis who did not achieve pregnancy (Group E.NP). Follicular fluid proteins were separated using 2D-electrophoresis,and their spots were compared, excised, and submitted to LC-ESI-MS/MS for proteins identification. The analysis showed 29 differentially expressed spots among the groups, and from these, 21 proteins were identified. Analysis showed some functional enrichment in the E.P group, including response to oxidative stress and apoptosis, while the E.NP group showed functions related to response to reactive oxygen species and positive regulation of apoptosis. These data suggest that endometriosis leads to differential protein expression in the follicular fluid, which can influences the outcome of pregnancy. These proteins may be potential targets for better diagnostics and new therapeutic intervention in affected women, as well as assisting in comprehending the physiopathologic mechanisms underlying endometriosis. Mol. Reprod. Dev. 80: 441-450, 2013. (c) 2013 Wiley Periodicals, Inc.806441450Center for Research in Urology, Human Reproduction Section, Sao Paulo Federal Universit