Dataset Information

Functional analysis of novel Rab GTPases identified in the proteome of 2 purified Legionella-containing vacuoles from macrophages

ABSTRACT: LCVs purified by immuno-magnetic separation and density gradient centrifugation (fraction 4) were resolved by 1D-SDS-PAGE, the gel lanes were excised in ten equidistant pieces and subjected to trypsin digestion. For the subsequent LC-MS/MS measurements, the digests were separated by reversed phase column chromatography using an EASYnLC apparatus with self-packed columns in a one-column setup. Following loading/ desalting in 0.1% acetic acid in water at a flow rate of 700 nL/min (maximum 220 bar), the peptides were separated by applying a binary non-linear gradient from 5-50% acetonitrile in 0.1% acetic acid over 70 min. The LC was coupled online to a LTQ-Velos Orbitrap mass spectrometer (Thermo Fisher, Bremen, Germany) at a spray voltage of 2.4 kV. After a survey scan in the Orbitrap (r = 30,000), MS/MS data were recorded for the twenty most intensive precursor ions in the linear ion trap. Singly charged ions were not taken into account for MS/MS analysis. The lock mass option was enabled throughout all analyses. After mass spectrometric measurement, MS data was converted into the mzxml format (version 3.1) by ReAdW version 4.3.1 and subsequently subjected to database searching via Sorcerer using Sequest (version 27, revision 11; SageN, Milptas, CA, USA) without charge state deconvolution and deisotoping performed. Database searching was performed either with a database of combined entries of L. pneumophila strain Philadelphia-1 (NC002942) and D. discoideum, or with a database of combined entries of L. pneumophila strain Philadelphia-1 and Mus musculus. Both databases were used as target/decoy databases with a list of common contaminants added (30739 and 64993 entries, respectively). Sequest was used assuming trypsinisation with a fragment ion mass tolerance of 1.00 Da and a search tolerance of 10 ppm for the overview scans. Oxidation of methionine was specified in Sequest as a variable modification. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR, USA) was used to filter and validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted when spectra exceeded Xcorr values of 2.2, 3.3 and 3.8 for doubly, triply and quadruply peptides with deltaCN values of more than 0.1. Protein identifications are based on at least 2 unique peptides. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to meet the principle of parsimony.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Legionella Pneumophila Dictyostelium Discoideum (slime Mold) Mus Musculus (mouse)

SUBMITTER: Andreas Otto

LAB HEAD: Andreas Otto

PROVIDER: PXD000647 | Pride | 2014-01-23

REPOSITORIES: Pride

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			Action	DRS
	120815_V2_P6_AO_ChH_Dicty_WT_1_1.mzXML	Mzxml
	120815_V2_P6_AO_ChH_Dicty_WT_1_10.mzXML	Mzxml
	120815_V2_P6_AO_ChH_Dicty_WT_1_2.mzXML	Mzxml
	120815_V2_P6_AO_ChH_Dicty_WT_1_3.mzXML	Mzxml
	120815_V2_P6_AO_ChH_Dicty_WT_1_4.mzXML	Mzxml

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Project description:Mtb was grown as either an actively growing normal culture or as a hypoxia-induced dormant culture. Whole cell lysates of live bacteria were obtained by mechanical disruption with silica beads. ATP-binding proteins were covalently labeled with desthiobiotin-tagged ATP (ActivX, Thermo Pierce). This chemoproteomic approach profiled the ATP-binding proteins present in either metabolic state. Labeled proteins were enriched via streptavidin affinity chromatography, digested with trypsin and subjected to tandem mass spectrometry (LC-MS/MS) for the identification of proteins containing a desthiobiotin modification of lysine (K +196 Da). Differential abundance of nucleotide binding proteins between the two growth conditions were quantified using label-free spectral counting and normalized spectral abundance factors (NSAF). DATABASE SEARCHING: Tandem mass spectra were extracted, charge state deconvoluted and deisotoped by Xcalibur version 2.2 SP1. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3.02) and SEQUEST (Thermo Fisher Scientific, San Jose, CA, USA; version v.27, rev. 11). Mascot and SEQUEST were set up to search the MtbReverse041712 database (7992 entries) assuming the digestion enzyme Trypsin. Parameters for both search engines were set to a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 2.5 Da. Oxidation of methionine, iodoacetamide derivative of cysteine and the desthiobiotin modification of lysine were specified in Mascot and SEQUEST as variable modifications. PROTEIN IDENTIFICATION AND LABEL-FREE QUANTITATION-- Scaffold (version Scaffold_3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they exceeded specific database search engine thresholds. Mascot identifications required ion scores to be greater than the associated identity scores and 50, 65, 65 and 65 for singly, doubly, triply and quadruply charged peptides. SEQUEST identifications required deltaCn scores of greater than 0.2 and XCorr scores of greater than 1.8, 2.0, 3.0 and 4.0 for singly, doubly, triply and quadruply charged peptides. Proteins identifications were accepted if they contained at least one identified peptide in at least two biological replicates. Peptide spectra meeting the most minimum requirement were manually inspected for quality. Quantification of proteins was performed on normalized spectral abundance factors for each protein (NSAF). BLAST-BASED SEQUENCE DESCRIPTION: The most relevant description for each of the sequences was acquired based on the significant BLAST results. The homologs for the sequences were retrieved using the Blastp algorithm and the nonredundant database of NCBI. The Blast2GO suite was used. GENE ONTOLOGY ANNOTATION: The Pfam domains were mapped to Gene Ontology (GO) terms using the lookup table provided by Pfam2go (http://www.geneontology.org/external2go/pfam2go). An in-house script was written to retrieve GO annotations based on the root term as "molecular function" and their distance from the root term. PFAM DOMAIN-BASED ANNOTATION: The InterPro and Pfam IDs corresponding to the GO term "ATP binding" (GO ID:0005524) were retrieved using the QuickGO (www.ebi.ac.uk/QuickGO/<http://www.ebi.ac.uk/QuickGO/>) and InterPro BioMart web services (http://www.ebi.ac.uk/interpro/biomart/martview). The ATP-binding associated domains were queried against the ATP-binding proteome data sets according to spectral quality (high, medium, low confidence). Their Pfam and InterPro descriptions, were identified using the InterProscan Web service, which was accessed via the Pipeline Pilot (Accelrys) implementation in the sequence analysis collection.

Project description:Cyanophora paradoxa muroplasts were isolated on a Percoll gradient. Muroplasts were fractionated into soluble (stroma), envelope, and thylakoid proteins. Protein samples from envelope membranes, thylakoids, stroma and total muroplasts were separated by 12% SDS-PAGE. Each Protein lanewas cut into 10 slices of variable size to match similar protein amounts. The gel slices were washed and diced to 1mm3 cubes. Gel pieces were destained by two consecutive washes with a 50:50 mixture of 200 mM ammonium bicarbonate and acetonitrile (ACN) for 20 min at 37°C. Proteins were reduced with 10mM DTT for 45 min at 56°C, followed by alkylation with 55mM iodoacetamide for 45 min at room temperature in the dark. After reduction and alkylation, gel pieces were washed twice with 100 mM ammonium bicarbonate, dehydrated with 100% ACN for 10 min, and dried in a SpeedVac. Gel pieces were fully covered in 50 mM ammonium bicarbonate containing 10ng/ml trypsin and rehydrated at 4°C. The samples were incubated overnight at 37°C and peptides in the supernatant were pooled after successive extraction steps using 50 mM ammonium bicarbonate, 100% ACN and 2.5% formic acid (FA), 50% ACN. The samples were dried in a SpeedVac and reconstituted in 5% ACN, 0.1% FA prior to MS analysis. Each of the samples were analysed in duplicates. Tryptic peptides of the thylakoid, envelope and stroma fractions were loaded onto a fritless 100 μm capillary packed in-house with 10cm of ProntoSIL C18 ace-EPS, 3µm. A gradient of 5-80% (v/v) ACN in 0.1% (v/v) FA was passed over the column with a duration of 115 min. The eluted peptides were directly electrosprayed into the LTQ orbitrap XL MS at a flow rate of 250 nl min-1 and a spray voltage of 1.3 kV. The instrument was operated in a 4-step data-dependent positive mode to identify the top three most abundant ions in a high-resolution MS scan (400 to 2000 m/z), which were then selected for fragmentation. MudPIT analyses were performed on the muroplast samples using an in-house packed analytical column consisting of 10 cm ProntoSIL C18 ace-EPS, 3µm (Bischoff) and 2 cm of a strong cationic exchange (SCX) material, 3µm in combination with an 6-step salt pulse gradient (0, 50, 100, 150, 200 and 500 mM ammonium acetate). Each salt-step peptides were eluted from the SCX to the C18-phase of the analytical column and eluated with a 130- min reverse-phase solvent gradient (5–80% ACN containing 0.1% FA). The eluate was directly electrosprayed into the LTQ orbitrap XL MS which was operated as described before. All MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search the C. paradoxa nuclear and muroplast protein database with a total of 32,286 entries, assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the C. paradoxa database also assuming trypsin. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0,80 Da and a parent ion tolerance of 10,0 ppm. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in Sequest and X! Tandem as variable modifications. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95,0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99,0% probability and contained at least 2 identified peptides.

Project description:Here we present the data obtained from a label-free quantitative proteomics analysis of soluble spinal cord extract derived from a mouse model of multiple sclerosis (EAE) and sham-induced mice. Samples were prepared offline using the FASP approach and then submitted for nano-LC-MS/MS analysis on an Orbitrap Velos instrument. After statistical evaluation of the data, 431 differentially expressed proteins (KS-test, p < 0.05) out of a total of ~1400 unique proteins were identified in the comparative spinal cord analysis (peptide FDR=0.55%).Database search and protein identification: Tandem mass spectra were extracted from .RAW files and searched using the SEQUEST-PVM v.27 (rev.9) (Eng et al., 1994) database program against a Mouse protein database downloaded as FASTA-formatted sequences from EBI-IPI (database version 3.72) which contains 56957 entries (with priority given to UniProt identifiers) as well as reverse decoy sequences to empirically assess the false identification rate. This search program was executed on a cluster computer to match the MS/MS spectra to the corresponding most highly correlated peptide sequences Mass tolerances for precursor (MS) and product ions (MS/MS) were set to 3 and 0 m/z, respectively. Searches were performed with the enzyme selectivity set to trypsin with one missed cleavage allowed and protein modifications included fixed carbamidomethylation of cysteines (57 Da). Match likelihoods were assigned a statistical confidence score using the STATQUEST probabilistic model (Kislinger et al., 2003) and candidate peptide identifications were filtered using an estimated peptide confidence score of ≥95%. A 10 ppm high accuracy mass filter accounting for isotopic shifts in the spectra was applied post-SEQUEST analysis thus improving the fidelity of protein identifications. Protein quantitation: To estimate relative protein levels, spectral counts were transformed into normalised spectral abundance factors (NSAF) as previously described (Mosley et al., 2009). Briefly, this involves dividing the spectral count (SC) of a protein by its length (Mw) and finally normalises this value to the sum of all SC/Mw.

Project description:Herein, proteomic profiling was conducted on susceptible sugarbeet infected with BNYVV to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugarbeet leaf tissue infected with BNYVV (beet necrotic yellow vein virus), quantified, and analyzed by mass spectrometry. A proprietary sugarbeet variety, KWS-rzNI, susceptible to all forms of BNYVV, was provided by KWS (Einbeck, Germany). To enable detection of a broad range of proteins associated with compatible interactions, two sources of BNYVV were used; standard BNYVV pathotype A, originally collected from Spence Field at the USDA-ARS in Salinas, CA, and resistance-breaking BNYVV-IV originating from the field, Rockwood 158, Imperial County, CA. Pooled leaf tissue from each cup was ground in liquid nitrogen and lyophilized. Total protein was extracted from 100 mg (dry weight) of lyophilized leaf tissue of each sample using the Plant Total Protein Extraction Kit (Sigma, St. Louis, MO) according to manufacturer’s recommendations. Protease inhibitors were added to liquid protein extracts (Pierce, Rockford, IL), and samples were stored at -80oC prior to analysis. Protein concentrations were determined via Bradford Assay [10] (Thermo Scientific, Rockford, IL) and 30 g of each sample underwent in-solution proteolytic digestion as previously described [11]. Briefly, samples were solubilized in 8 M urea, 0.2% Protease Max (Promega, Madision, WI), then reduced with dithiothreitol, alkylated with iodoacetamide, and digested with 1% Protease Max and trypsin at 37oC for 3 hours. Samples were dried in a Speed Vac® vacuum centrifuge, desalted using Pierce PepClean C18 spin columns (Pierce, Rockford, IL), dried and resuspended in 30 ull 3% ACN, 0.1% formic acid. All solvents, water, and acid were LC-MS/MS grade from Sigma (St. Louis, MO). 1 l of each sample (three replicates from each soil type) was analyzed in duplicate injections via LC-MS/MS . Peptides were purified and concentrated using an on-line enrichment column (Thermo Scientific 5m, 100 m ID x 2 cm C18 columns). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Thermo Scientific EASYnano-LC, 3m, 75 m ID x 100 mm C18 column) using a 90 minute linear gradient from 10%-30% buffer B (100% ACN, 0.1% formic acid) at a flow rate of 400 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific Orbitrap Velos Pro) and spectra collected over a m/z range of 400-2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific) with a S/N threshold of 1.5 and 1 scan/group. MS/MS spectra were searched against a Uniprot Amaranthacae database (3,645 entries, version 03/06/13) concatenated to a reverse database and the Uniprot Mus musculus database (50,665 entries, version 03/06/13) using the Mascot database search engine (Matrix Science, version 2.3.02) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research). Due to the limited size of the Amaranthaceae database, the Mus musculus sequences were added to ensure adequate database size for statistical scoring and calculation of false discovery rates. The following search parameters were used in Mascot: monoisotopic mass, parent mass tolerance of 20 ppm, fragment ion mass tolerance of 0.8 Da , complete tryptic digestion allowing two missed cleavages, variable modification of methionine oxidation, and a fixed modification of cysteine carbamidomethylation. SEQUEST search parameters were the same except for a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 0.0120 Da. Peptide identifications from both of the search engines were combined using probabilistic protein identification algorithms in Scaffold 4 [13, 14] (Version 4.0.3, Proteome Software, Portland, OR) with protein clustering enabled. All data files were then combined using the “mudpit” option in Scaffold 4 generating a composite listing for all proteins identified. Thresholds were set to 99% protein probability, 95% peptide probability, and a 2 unique peptide minimum was required. The peptide FDR was 0% after manual validation of all proteins identified by 2 unique peptides [15]. Criteria for manual validation included the following: 1) a minimum of at least 5 theoretical y or b ions in consecutive order that are peaks greater than 5% of the maximum intensity; 2) an absence of prominent unassigned peaks greater than 5% of the maximum intensity; and 3) indicative residue specific fragmentation, such as intense ions N-terminal to proline and immediately C-terminal to aspartate and glutamate.

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Functional analysis of novel Rab GTPases identified in the proteome of 2 purified Legionella-containing vacuoles from macrophages

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