Jerusa Simone Garcia

Trends in metal-binding and metalloprotein analysis

This review describes recent tendencies for metal-binding and metalloprotein analysis, emphasizing metal quantification in proteins through X-ray, atomic absorption, mass spectrometric techniques, and others. Hyphenated techniques such as capillary electrophoresis-synchrotron radiation X-ray fluorescence (CE-SRXRF), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF-MS), etc. are also presented. As protein separation techniques electrophoresis (mainly sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are indicated, due to their inherent sensitivity, resolution and/or easy implementation. Latest challenges in metallomics are also commented.

Single embryo and oocyte lipid fingerprinting by mass spectrometry

Methods used for lipid analysis in embryos and oocytes usually involve selective lipid extraction from a pool of many samples followed by chemical manipulation, separation and characterization of individual components by chromatographic techniques. Herein we report direct analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of single and intact embryos or oocytes from various species. Biological samples were simply moisturized with the matrix solution and characteristic lipid (represented by phosphatidylcholines, sphingomyelins and triacylglycerols) profiles were obtained via MALDI-MS. As representative examples, human, bovine, sheep and fish oocytes, as well as bovine and insect embryos were analyzed. MALDI-MS is shown to be capable of providing characteristic lipid profiles of gametes and embryos and also to respond to modifications due to developmental stages and in vitro culture conditions of bovine embryos. Investigation in developmental biology of the biological roles of structural and reserve lipids in embryos and oocytes should therefore benefit from these rapid MALDI-MS profiles from single and intact species.

Secretome of the preimplantation human embryo by bottom-up label-free proteomics

AbstractA bottom-up label-free mass spectrometric proteomic strategy was used to analyse the protein profiles of the human embryonic secretome. Culture media samples used for embryonic culture of patients undergoing intracytoplasmic sperm injection cycles were selected as a test case for this exploratory proof-of-principle study. The media were stored after embryo transfer and then pooled into positive (n = 8) and negative (n = 8) implantation groups. The absolute quantitative bottom-up technique employed a multidimensional protein identification technology based on separation by nano-ultra-high pressure chromatography and identification via tandem nano-electrospray ionization mass spectrometry with data-independent scanning in a hydrid QqTOF mass spectrometer. By applying quantitative bottom-up proteomics, unique proteins were found exclusively in both the positive- and negative-implantation groups, which suggest that competent embryos express and secrete unique biomarker proteins into the surrounding culture medium. The selective monitoring of these possible secretome biomarkers could make viable procedures using single-embryo transfer. FigureA bottom-up label-free proteomic mass spectrometric analysis of the human embryo secretome is described. This approach seems to allow quantification and identification of unique proteins related to positive- and negative-implantation groups, which can be further validated as biomarkers for selection and transfer of a single embryo.

Detection and expression analysis of recombinant proteins in plant‐derived complex mixtures using nanoUPLC‐MSE

The use of mass spectrometry to identify recombinant proteins that are expressed in total soluble proteins (TSPs) from plant extracts is necessary to accelerate further processing steps. For example, the method consists of TSP sample preparation and trypsin digestion prior to the preliminary characterization using nanoUPLC-MS(E) analysis of the recombinant proteins that are expressed in TSP samples of transgenic soybean seeds. A TSP sample as small as 50 μg can be effectively analyzed. In this study, transgenic soybean seeds that expressed recombinant cancer testis antigen (CTAG) were used. The procedure covered 30% of the protein sequence and was quantified at 0.26 ng, which corresponded to 0.1% of the TSP sample. A comparative proteomic profile was generated by the comparison of a negative control and sample that showed a unique expression pattern of CTAG in a transgenic line. The experimental data from the TSP extraction, sample preparation and data analysis are discussed herein.

Evaluation of metal-ion stress in sunflower (Helianthus annuus L.) leaves through proteomic changes

In this work, sunflowers (Helianthus annuus L.) were cultivated using soil and vermicompost as substrate, and plant irrigation was carried out using either a Zn solution or a mixed ions solution (Cd, Cu, Pb and Zn). After plant harvesting, the effects of metal-ion contamination on proteins expression (either up- or down-regulation) in sunflower leaves were evaluated using two-dimensional electrophoresis (2-DE), gel images and mass spectrometry (MALDI-QTOF MS). When Zn or mixed ions solution was added to the substrate, nine proteins showed different expressions. Another twenty-three protein spots also showed considerable variation when both treatments (Zn or mixed ions) were applied. Twelve of these proteins were successfully characterized, six of them being reported for the first time in Helianthus annuus L. Two other proteins showed new sequences that have been downloaded to the protein databank.

Purification and structural characterization of fengycin homologues produced by Bacillus subtilis LSFM-05 grown on raw glycerol.

Raw glycerol is a byproduct of biodiesel production that currently has low to negative value for biodiesel producers. One option for increasing the value of raw glycerol is to use it as a feedstock for microbial production. Bacillus subtilis LSFM 05 was used for the production of fengycin in a mineral medium containing raw glycerol as the sole carbon source. Fengycin was isolated by acid precipitation at pH 2 and purified by silica gel column chromatography and characterized using electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) with collision-induced dissociation (CID). The mass spectrum revealed the presence of the ions of m/z 1,435.7, 1,449.9, 1,463.8, 1,477.8, 1,491.8 and 1,505.8, which were further fragmented by ESI-MS/MS. The CID profile showed the presence of a series of ions (m/z 1,080 and 966) and (m/z 1,108 and 994) that represented the different fengycin homologues A and B, respectively. Fengycin homologues A and B are variants that differ at position 6 of the peptide moiety, having either Ala or Val residues, respectively. Mass spectrometry analyses identified four fengycin A and three fengycin B variants with fatty acid components containing 14–17 carbons. These results demonstrate that raw glycerol can be used as feedstock to produce fengycin, and additional work should focus on the optimization of process conditions to increase productivity.

Fast Screening and Secure Confirmation of Milk Powder Adulteration with Maltodextrin via Electrospray Ionization−Mass Spectrometry [ESI(+)−MS] and Selective Enzymatic Hydrolysis

Direct-infusion electrospray ionization-mass spectrometry [ESI(+)-MS] of several milk powder samples, confiscated by the Brazilian Federal Police, showed ions accounting for sodiated and potassiated molecules of disaccharides (m/z 365 and 381) as well as trisaccharides (m/z 527 and 543), whereas monosaccharide ions were not detected. The trisaccharide ions were not detected in samples of genuine milk powder, raising the suspicion that their presence indicates adulteration by the addition of maltodextrin. In control samples, maltose and maltotriose were hydrolyzed by alpha-glucosidase and not beta-galactosidase, whereas lactose was resistant to alpha-glucosidase but was hydrolyzed with beta-galactosidase. Samples suspected of being adulterated behaved in the same fashion, confirming the presence of maltose and maltotriose or maltodextrin. Direct-infusion ESI-MS is shown therefore to provide rapid screening of milk powder for adulteration with maltodextrin, whereas its combination with selective enzymatic hydrolysis provides highly reliable confirmation for unambiguous results.

Effect of endometriosis on the protein expression pattern of follicular fluid from patients submitted to controlled ovarian hyperstimulation for in vitro fertilization

BACKGROUND The aim of this study was to evaluate protein expression profile and quantify the proteins present in follicular fluid (FF) samples from women with endometriosis and pregnant women without endometriosis. METHODS A prospective case-control study was carried out including women with Stage III or IV endometriosis (Group I) and pregnant women without endometriosis (Group II), both at the maximum age of 35 years. Women were submitted to controlled ovarian stimulation for in vitro fertilization, and FF was collected after ultrasound-guided ovarian aspiration. FF from both ovaries was pooled, and patient samples were pooled according to Group I or II. Pooled protein samples were separated and analyzed by MudPIT (multidimensional protein identification technology followed by Expression(E) and label-free quantification with ProteinLynxGlobalServer 2.4v, Identity(E) and Expression(E) software). RESULTS A total of 416 proteins or randomic sequence were identified, 62 proteins differentially expressed between Groups I and II. One (1.6%) was expressed at a higher level and 36 (58.1%) were uniquely expressed in Group I, whereas 8 (12.9%) were expressed at a higher level and 17 (27.4%) were uniquely expressed in Group II. Of all these, 15 (24.2%) are related to binding, 1 (1.6%) to immune response, 8 (12.9%) to cell division, 3 (4.8%) to cellular metabolism, 16 (25.8%) to general function and 19 (30.6%) do not yet present an identified function. CONCLUSIONS Protein expression profiles of patients with and without endometriosis identified at least 64 proteins differentially expressed, which may be related to the physiopathology of endometriosis. These proteins may additionally be useful in determining potential biomarkers for diagnostics, as well as for therapeutic intervention in women with infertility due to endometriosis.

DIFFERENTIAL SEMINAL PLASMA PROTEOME ACCORDING TO SEMEN RETRIEVAL IN MEN WITH SPINAL CORD INJURY

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 (MS(E)) and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE). RESULT(S) A total of 638 different proteins were identified by MS(E) 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.

Thermospray generation directly into a flame furnace—An alternative to improve the detection power in atomic absorption spectrometry

Recent developments and applications in the production of thermosprays directly into flame furnaces to improve the analytical sensitivity in atomic absorption spectrometry are reviewed in this manuscript. Principles, characteristics, instrumentation, and applications of this analytical technique for trace elements determination in several matrices are discussed. The use of preconcentration procedures to allow low detection limits for ultra-trace levels using TS-FF-AAS is presented and current perspectives and future trends of this technique are also discussed.