Proteomic Profile of multidrug- resistant Serratia marcescens under meropenem challenge
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Synapt MS
ORGANISM(S): Serratia Marcescens
SUBMITTER: 
Celia Soares
LAB HEAD: Dayane Moraes
PROVIDER: PXD059425 | Pride | 2025-01-03
REPOSITORIES: Pride
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Project description:Fungal diseases significantly impact human mortality and morbidity. However, despite the number of annual deaths caused by fungi exceeding 1.6 million, these microorganisms are often neglected. Fonsecaea pedrosoi is the main etiologic agent of chromoblastomycosis (CBM), a chronic mycosis that affects the skin and subcutaneous tissue that, in 2017, was included in the WHO list of neglected tropical diseases. It is essentially an occupational disease that affects individuals in poverty, causing significant morbidity and mortality. CBM is a disease of global distribution, and most cases are described in tropical and subtropical regions of America, Africa and Asia. Despite its importance in the etiology of the disease, the molecular mechanisms involved in the interaction of F. pedrosoi with the host are still poorly explored. During the infectious process, the host limits the availability of zinc to fungal pathogens in order to contain the infection, a process called nutritional immunity. In this work, we show that F. pedrosoi is able to grow in a wide range of zinc concentration and that genes related to zinc uptake are induced in zinc-limiting conditions. Proteomic analysis revealed that after 48 h of exposure to zinc scarcity synthesis of fatty acid and the pentose-phosphate pathway are up-regulated. On the other hand, protein synthesis is repressed. Additionally, glucan increases while chitin content is reduced in the cell wall under zinc limitation, demonstrating that cell wall remodeling is important for adaptation to this stress condition.
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Project description:Supernatant and precipitate protein fractions of human bronchial smooth muscle cells (HBSMC) after sonication and centrifugation were analyzed using 1D SDS-PAGE. The gel lanes were cube-cut into thirty-three 1.1 mm x 1.1 mm x 1 mm (gel thickness) squares and each gel piece was subjected to in-gel digestion and quantitative LC-MS/MS. Totally 2552 proteins were assigned from the lane of supernatant and 2614 protein species from the lane of precipitate. The results are being further analyzed, e.g. 1) for the comparison between the proteins species residing in the two fractions, and 2) for correlation with the nondenaturing 2DE-grid cut-quantitative LC-MS/MS results (Jin, et al. Electrophoresis 2015, 36, 1711-1723, Jin, et al. Electrophoresis 2015, 1991-2001, PRIDE project PXD001471).
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Project description:We applied a non targeted mass spectrometric assay (LCMSE), to quantify 55 abundant plasma proteins in specimens from 31 healthy volunteers. Quantitation was done by HI3 peptide measurement using a single internal standard to estimate protein concentrations. Results for 7 apolipoproteins were compared with those obtained using isotope labelled standards, while 12 proteins were compared to routine immunoassays. Blood samples were obtained from 31 healthy volunteers (17 males; 14 females) with a median age of 46 years and a range of 22-67 years. Total plasma protein concentration was assayed with a BCA-assay (20) according to the manufacturer’s protocol (Thermo). Samples were diluted tenfold in 0.1% Rapigest SF (Waters Corporation, Milford, MA), 50 mM ammonium bicarbonate and heated at 95° C for 15 min. Subsequently, plasma samples were reduced with 5 mM dithiothreitol (60° C, 30 min) and alkylated with 15 mM iodoacetamide (ambient temperature, dark, 30 min). Proteolytic digestion was performed with modified trypsin (gold grade, Promega, Madison WI) at 0.3 units/µg protein, (37° C, 20 hours) unless indicated otherwise. Following digestion, Rapigest SF was broken down by adding 1% trifluoroacetic acid (pH<2, 37 °C, 45 min). Peptide solutions were centrifuged (20,000 x g, 10 min) and supernatant was collected. Prior to analyses a MASSPREP protein digestion standard (Waters Corporation, ADH1 or ENO from Saccharomyces cerevisiae) was added for quantitation purposes. LC-MS analyses were performed using ~ 0.21 µg of the final plasma protein digest mixtures (384 times total dilution) unless indicated otherwise. LC separations of tryptic peptides were performed with a NanoAcquity system (Waters Corporation), coupled to a Synapt G2 quadrupole time of flight mass spectrometer (Waters Corporation, Manchester, UK). Accurate mass precursor and fragment ion LC-MS data were collected in data independent LCMSE mode of acquisition. Continuum LC-MS data were processed using ProteinLynx GlobalSERVER version 2.5 (PLGS 2.5, Waters Corporation). Parameter settings: digest reagent trypsin, allow 1 ‘missed cleavage’, search tolerances automatic, typically 5 ppm for precursor and 15 ppm for product ions, fixed modification cysteine carbamidomethylation, and variable modification methionine oxidation. Protein identifications were obtained searching the human SwissProt entries of an UniProt database (release 13.2). This database was modified to include N-terminal processing of proteins using protein maturation device software, with ADH1 and ENO1 of S. cerevisiae appended as internal standard to address technical variation and allow concentration determinations. Estimation of false-positive identification rates was done by searching a randomized version of the abovementioned human protein database generated within PLGS 2.5. Data were exported as csv-files for further, detailed analysis. Stringent criteria were applied for quantitation, protein identifications were only considered significant if reported in 14 or more samples. Protein false positive identification rates were estimated using the criteria mentioned above and no false positives were identified in these searches. DATASET: KC1-31: 31 healthy volunteers QC1-10: 10 times injection of a single sample to ascertain analytical variation over 9 days of measurements. 20120802_QC1-QC6: 6 pooled plasma samples worked up and analysed during a single day of measurements to ascertain Intra-assay variation. 20120427_S2,S4;20120525_S18,S19;20120626_S3,S4;20120802_QC7: 7 pooled plasma samples processed and analysed during 3 months of routine measurements to analyse Inter-assay variation 20130416_s1-s5: pooled plasma samples spiked with a QCONCATAMER for 11 apolipoproteins labelled by heavy Lysine and Arginine (+6.02 AMU) to quantify apolipoproteins by internal isotope labelled standard.
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