Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

Follicular fluid alterations in endometriosis: label-free proteomics by MS(E) as a functional tool for endometriosis.

Endometriosis is a chronic gynecological condition that affects 10-32% of women of reproductive age and may lead to infertility. The study of protein profiles in follicular fluid may assist in elucidating possible biomarkers related to this disease. For this, follicular fluid samples were obtained from women with tubal factor or minimal male factor infertility who had pregnancy outcomes after in vitro fertilization (IVF) treatment (control group, n = 10), women with endometriosis (endometriosis group, n = 10), along with the endometrioma from these same patients were included (endometrioma group, n = 10). For proteomic analysis, samples were pooled according to their respective groups and normalized to protein content. Proteins were analyzed by in tandem mass spectrometry (MS(E)) Spectra processing and the ProteinLynx Global Server v.2.5. was used for database searching. Data was submitted to the biological network analysis using Cytoscape 2.8.2 with ClueGO plugin. As a result, 535 proteins were identified among all groups. The control group differentially or uniquely expressed 33 (6%) proteins and equal expression of 98 (18%) proteins was observed in the control and endometriosis groups of which 41 (7%) proteins were further identified and/or quantified. Six (1%) proteins were observed in both the endometriosis and endometrioma groups, but 212 (39%) proteins were exclusively identified and/or quantified in the endometrioma group. There were 9 (1%) proteins observed in both the control and endometrioma groups and there were 139 (25%) proteins common among all three groups. Distinct differences among the protein profiles in the follicular fluid of patients included in this study were found, identifying proteins related to the disease progression and IVF success. Thus, some pathways related to endometriosis are associated with the presence of specific proteins, as well as the absence of others. This study provides a first step to the development of more sensitive diagnostic tests and treatment.

Chemo-, regio- and stereoselective Heck arylation of allylated malonates: mechanistic insights by ESI-MS and synthetic application toward 5-arylmethyl-γ-lactones.

We describe herein a general method for the controlled Heck arylation of allylated malonates. Both electron-rich and electron-poor aryldiazonium salts were readily employed as the aryl-transfer agents in good yields and in high chemo-, regio-, and stereoselectivity without formation of decarboxylated byproducts. Reaction monitoring via ESI-MS was used to support the formation of chelated Pd species through the catalytic cycle. Additionally, some Heck adducts were successfully used in the total synthesis of pharmacologically active γ-lactones.

Hyaluronidase alters the lipid profile of cumulus cells as detected by MALDI-TOF MS and multivariate analysis.

This research aimed to study the changes in lipid composition in cumulus cells using hyaluronidase according to the intracytoplasmic sperm injection protocol commonly used in human reproduction clinics. The lipid extraction was performed by the Blight-Dyer protocol and the lipid profiles were obtained by MALDI-TOF MS in positive and negative modes. The mass spectra data were processed with MassLynx and the statistical analysis was performed using MetaboAnalyst 2.0. Fifteen ions were selected for each mode as potential markers for differences between the groups. These ions were identified in the human metabolome database as phosphatidylserine with and without treatment, phosphatidylethanolamine in the after treatment group and phosphatidylinositol in the before treatment group, which are lipids that may be involved in cell apoptosis and signaling. We concluded that MALDI-TOF MS coupled with multivariate analysis can be utilized as a strategy to obtain and study the lipid profiles of cumulus cells and as a tool to study the metabolic state of cumulus cells.

On the mechanism of the aza-Morita-Baylis-Hillman reaction: ESI-MS interception of a unique new intermediate.

Solutions of aza-Morita-Baylis-Hillman (aza-MBH) reactions were directly monitored by ESI(+)-MS(/MS) spectrometry to obtain information on their mechanism. A unique bis-sulfonamide intermediate was intercepted and characterized and, based on this novel species, a mechanism that rationalizes the uniqueness of aza-MBH reactions is proposed.

Venturi easy ambient sonic-spray ionization.

The development and illustrative applications of an ambient ionization technique termed Venturi easy ambient sonic-spray ionization (V-EASI) is described. Its dual mode of operation with Venturi self-pumping makes V-EASI applicable to the direct mass spectrometric analysis of both liquid (V(L)-EASI) and solid (V(S)-EASI) samples. V-EASI is simple and easy to assemble, operating solely via the assistance of a sonic stream of nitrogen or air. The sonic gas stream causes two beneficial and integrated effects: (a) the self-pumping of solutions via the Venturi effect and (b) sonic-spray ionization (SSI) of analytes either in solution or resting on solid surfaces. In its liquid mode, V(L)-EASI is applicable to analytes in solution, forming negatively and/or positively charged intact molecular species in a soft fashion with little or no fragmentation. In its solid mode, V(S)-EASI relies on Venturi self-pumping of a proper SSI solvent solution in combination with SSI to form a stream of bipolar charged droplets that bombard the sample surface, causing desorption and ionization of the analyte molecules. As for its precursor technique (EASI), V-EASI generates bipolar droplets with considerably lower average charging, which increases selectivity for ionization with high signal-to-noise ratios and clean spectra dominated by single molecular species with minimal solvent ions. V-EASI also operates in a voltage-, heat-, and radiation-free fashion and is therefore free of thermal, electrical, or discharge interferences.