{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP5_FIND170_A.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP1_FIND22.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP4_FIND150lower.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP5_FIND170_B.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141110_JMK_AP041_MALP.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP2_FIND91.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP3_FIND150upper.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141110_JMK_AP041_WALP.raw"],"Mgf":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP1_FIND22.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP5_FIND170_A.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP3_FIND150upper.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141110_JMK_AP041_WALP.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP4_FIND150lower.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP5_FIND170_B.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141110_JMK_AP041_MALP.mgf","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/141212_JMK_AP2_FIND91.mgf"],"Other":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F045557.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F034618.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F034623.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F034610.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F045558.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F034625.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F034627.dat","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/06/PXD004133/F034629.dat"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":{"citationCount":0,"reanalysisCount":0,"viewCount":244,"searchCount":0},"additional":{"labhead_mail":["parret@embl-hamburg.de"],"submitter":["Joanna Kirkpatrick"],"technology_type":["Mass Spectrometry","Gel-based experiment"],"disease":["Tuberculosis"],"software":["Not available"],"submitter_keywords":["Diagnostic marker","Mycobacterium tuberculosis","Protein production"],"full_dataset_link":["http://www.ebi.ac.uk/pride/archive/projects/PXD004133"],"sample_protocol":["A standardized procedure for generating recombinant proteins from Mycobacterium tuberculosis (Mtb), for use in clinical applications, was developed. Proteins were obtained from Mycobacterium smegmatis and purification using chromatography (Ni-NTA purification followed by size exclusion chromatography (SEC). When required, either ion exchange (IEX) or hydrophobic interaction (HIC) chromatography was also used to improve protein purity. The potential of the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. Protein purity was assessed both by size exclusion chromatography and mass spectrometry. Briefly, the fractions containing the target proteins were subjected to intact protein MW analysis. Samples were acidified prior to injection onto the Acquity UPLC System (Waters GmbH, Eschborn, Germany). Approximately 3-4 µg protein were loaded onto a Waters ACQUITY UPLC Protein BEH C4 column (300 Å, 1.7 µm, 2.1 mm X 150 mm coupled directly to a Q-Tof Premier mass spectrometer (Waters) using the standard ESI source in positive ion mode. Solvent A was water, 0.1% formic acid and solvent B was acetonitrile, 0.1% formic acid. The samples were loaded in 96% A, 4% B (0.2 mL/min). The column was held at 4% B for 5 min before ramping to 25% B by 6 min. A linear gradient to 80% B was then applied until 17 min. For the MS, a spray voltage of 3.5 kV was applied, cone voltage of 35 V and extraction cone at 5 V. Desolvation temperature was set at 350 °C, with source temperature 120 °C. Desolvation gas was nitrogen (600 L/min). Collision energy was 5 eV with argon in the collision cell (5.3 e-3 mbar). Data were acquired in continuum mode from 500-3500 m/z. The instrument was externally calibrated with NaI, against which a reference standard of intact myoglobin was checked immediately prior to sample data acquisition. Additionally, the samples were either in-solution digested or the fractions were loaded on an SDS-PAGE gel and then subjected to in-gel enzymatic digestion as follows: Protein samples in solution were taken and digested with either trypsin (Promega), WaLP or MaLP (Sigma Aldrich); wild type α-lytic protease (WaLP) and an active site mutant of WaLP, M190A α-lytic protease (MaLP) enzymes [4]. All reagents used were dissolved in 50 mM ammonium bicarbonate. Approximately 20 µg of protein was taken and diluted to 0.25 µg/µL in 50 mM ammonium bicarbonate. Proteins were then reduced (2.5 µL of 50 mM DTT, 56 °C, 30 minutes) and alkylated (110 mM iodoacetamide, 2.5 µL, room temperature, in the dark, 20 minutes). The enzymatic digestion (0.5 µL of a 1 µg/µL enzyme solution) was carried out overnight at 37 °C. For the trypsin digestion of gel bands, gel pieces were cut into 1 mm cubes for preparation prior to in-gel digestion. Reagents were prepared in 100 mM ammonium bicarbonate. The gel pieces reduced (DTT, 56 °C, 30 minutes, 10 mM) and alkylated (iodoacetamide, room temperature, in the dark, 20 minutes, 55 mM). Trypsin digestion (1 ng/µL trypsin solution) proceeded overnight at 37 °C. Peptides were extracted and analysed by LC-MS/MS as follows: Peptides were separated using the nanoAcquity UPLC system (Waters) fitted with a trapping (nanoAcquity Symmetry C18, 5µm, 180 µm x 20 mm) and an analytical column (nanoAcquity BEH C18, 1.7µm, 75µm x 200mm). The outlet of the analytical column was coupled directly to an LTQ Orbitrap Velos Pro (Thermo Fisher Scientific GmbH, Bremen, Germany) using the Proxeon nanospray source. Solvent A was water, 0.1 % formic acid and solvent B was acetonitrile, 0.1 % formic acid. The samples (100 fmol) were loaded in solvent A, at 5 µL/min, onto the trapping column, for 6 minutes. Peptides were eluted via the analytical column at 0.3 µL/min. During the elution step, the percentage of solvent B increased in a linear fashion from 3 % to 40 % in 15 minutes. The peptides were introduced into the mass spectrometer via a Pico-Tip Emitter 360 µm OD x 20 µm ID; 10 µm tip (New Objective, Inc., Woburn, USA) and a spray voltage of 2.2 kV was applied. The capillary temperature was set at 300 °C. Full scan MS spectra (300-1700 m/z) were acquired in profile mode in the FT with resolution of 30000. The filling time was set at maximum of 500 ms with limitation of 106 ions. The most intense ions (up to 15) from the full scan MS were selected for fragmentation in the LTQ. Normalized collision energy of 40 % was used, and the fragmentation was performed after accumulation of 3 x 104 ions or after filling time of 100 ms for each precursor ion (whichever occurred first). MS/MS data were acquired in centroid mode. Only multiply charged (2-4+) precursor ions were selected for MS/MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients."],"repository":["Pride"],"quantification_method":["Not available"],"modification":["iodoacetamide derivatized residue","monohydroxylated residue"],"data_protocol":["Mass spectrometry: For the intact protein molecular weight analysis. Spectra from the chromatogram protein peak were then summed and intact mass was calculated using the MaxEnt1 maximum entropy algorithm (Waters) to give the zero charge deconvoluted molecular weight. For the in-solution or in-gel digested samples, MSConvert (Proteowizard) was used for creating .mgf files from the raw data, needed for searching in MASCOT version 2.2.07 (Matrix Science Ltd, London, UK). The data were searched against the NCBInr (bacteria) database or an in-house user database to which the expected sequences had been appended. Both databases contain a list of common contaminants. The data were searched with the following modifications: Carbamidomethyl (C) (Fixed) and Oxidation (M) (Variable). The mass error tolerance for the full scan MS spectra was set at 20 ppm and for the MS/MS spectra at 0.5 Da. Data were searched with one missed cleavage for tryptic digest data. For WaLP and MaLP enzymes, no enzyme specificity was selected with 0 missed cleavages. Results were processed using Proteinscape 2.1 (Bruker Daltonik GmbH, Bremen, Germany). Mascot score cut-offs of 20 (peptide) and 40 (protein) were used."],"omics_type":["Proteomics"],"labhead":["Annabel Parret"],"instrument_platform":["LTQ Orbitrap Velos"],"labhead_affiliation":["Head of the Molecular Biology and Biochemistry Laboratory EMBL Hamburg Notkestrasse 85, 22607 Hamburg, Germany"],"submission_type":["PARTIAL"],"species":["Mycobacterium Tuberculosis H37rv"],"submitter_mail":["joanna.kirkpatrick@leibniz-fli.de"],"publication":["27400835 Milewski MC, Broger T, Kirkpatrick J, Filomena A, Komadina D, Schneiderhan-Marra N, Wilmanns M, Parret AH. A standardized production pipeline for high profile targets from Mycobacterium tuberculosis. Proteomics Clin Appl. 2016 10(9-10):1049-1057 10.1002/prca.201600033"],"curator_keywords":["Biological","Biomedical"],"submitter_affiliation":["The Francis Crick Institute"],"submitter_country":["United Kingdom"],"pubmed_abstract":["