Proteomic analysis of ARF regulatory molecules co-immunoprecipitating with human SDC4 (huSDC4)
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Mark Morgan
PROVIDER: MSV000093211 | MassIVE | Fri Oct 27 15:27:00 BST 2023
REPOSITORIES: MassIVE
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Project description:S-nitrosoproteome of human unterine smooth muscle in different states of pregnancy (Labor, non labor, and preterm labor). Samples were isolated by the biotin switch and streptavidin pulldown before being analyzed by MS/MS on an Orbitrap instrument. Database Searching and bioinformatics: Tandem mass spectra were extracted; charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, version v.27, rev. 11). Sequest was initiated to search the database containing ipi.HUMAN.v3.87, the Global Proteome Machine cRAP v.2012.01.01, and random decoy sequences (183158 entries) assuming the digestion enzyme trypsin. Sequest was searched with a fragment ion mass tolerance of 1.00 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine, iodoacetamide derivative of cysteine, and n-ethylmaleimide on cysteine were specified in Sequest as variable modifications. Criteria for Protein Identification-PROTEOIQ (V2.6, www.nusep.com) was used to validate MS/MS based peptide and protein identifications. Peptides were parsed before analysis with a minimum Xcorr value of 1.5 and a minimum length of 6 amino acids. There were no matches to the concatenated decoy database and therefore a false discovery value is not applicable or "0". 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 95.0% probability and contained at least two identified peptides with 5 spectra per peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
2014-03-02 | PXD000226 | Pride
Project description:1D-LC-MS/MS analysis of OGE-prefractionated and TMT labeled mouse samples consiting of 2 unstimulated and 2 stimulated T-cell samples. The acquired raw-files were converted to the mascot generic file (mgf) format using the msconvert tool (part of ProteoWizard, version 3.0.4624 (2013-6-3)). Using the MASCOT algorithm (Matrix Science, Version 2.4.0), the mgf files were searched against a decoy database containing normal and reverse sequences of the predicted SwissProt entries of mus musculus (www.ebi.ac.uk, release date 16/05/2012) and commonly observed contaminants (in total 33,832 sequences) generated using the SequenceReverser tool from the MaxQuant software (Version 1.0.13.13). The precursor ion tolerance was set to 10 ppm and fragment ion tolerance was set to 0.01 Da. The search criteria were set as follows: full tryptic specificity was required (cleavage after lysine or arginine residues unless followed by proline), 2 missed cleavages were allowed, carbamidomethylation (C), TMT6plex (K and peptide n-terminus) were set as fixed modification and oxidation (M) as a variable modification. Next, the database search results were imported to the Scaffold Q+ software (version 4.1.1, Proteome Software Inc., Portland, OR) and the protein false identification rate was set to 1% based on the number of decoy hits. Specifically, peptide identifications were accepted if they could be established at greater than 94.0% probability to achieve an FDR less than 1.0% by the scaffold local FDR algorithm. Protein identifications were accepted if they could be established at greater than 6.0% probability to achieve an FDR less than 1.0% and contained at least 1 identified peptide. Protein probabilities were assigned by the Protein Prophet program (Nesvizhskii, et al, Anal. Chem. 2003; 75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. For quantification, acquired reporter ion intensities in the experiment were globally normalized across all acquisition runs. Individual quantitative samples were normalized within each acquisition run. Intensities for each peptide identification were normalized within the assigned protein. The reference channels were normalized to produce a 1:1 fold change. All normalization calculations were performed using medians to multiplicatively normalize data. A list of identified and quantified proteins is available in the xls file.
2014-12-02 | PXD000543 | Pride
Project description:IP-LC/MS pulldown assay for identifying TFPI2-associated membrane protein in human primary microglia.
Tandem mass spectra were extracted. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.8.0). Mascot was set up to search the uniprot-SP-human_20190906_20200629 database (20432 entries) assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.050 Da and a parent ion tolerance of 10.0 PPM. Carbamidomethyl of cysteine was specified in Mascot as a fixed modification. Deamidated of asparagine and glutamine, oxidation of methionine and acetyl of the n-terminus were specified in Mascot as variable modifications.
Scaffold (version Scaffold_5.1.2, 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 90.0% probability by the Peptide Prophet algorithm (Keller, A et al Anal. Chem. 2002;74(20):5383-92) with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, Al et al Anal. Chem. 2003;75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
Sample #3 is protein eluted from the complex containing TFPI2 antibody. Sample #4 is IgG control.
2022-10-31 | MSV000090624 | MassIVE
Project description:The loss of von Hippel-Lindau (VHL) protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2alpha (HIF2alpha) instead of hypoxia-inducible factor-1alpha (HIF1alpha). MS/MS data generated by data dependent acquisition via the LTQ-FT were extracted by BioWorks version 3.3 and searched against a composite IPI human protein sequence database (IPI _human_v 3.73.par) containing both forward and randomized sequences using Mascot version 2.2 (Matrix Science, Boston, MA) search algorithm. Mascot was searched with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 15.0 ppm assuming trypsin as the digestion enzyme with the possibility of one missed cleavage. Carbamidomethylation of cysteine was specified as a fixed modification, while oxidation of methionine, N-terminal protein acetylation, N-terminal peptide and lysine carbamoylation, and phosphorylation of serine and threonine were specified as variable modifications. The analysis of VHL-wt (Caki-2) human RCC and VHL-mut (786-O) cells was carried out from three independent, biological samples from each cell line (tryptic digests of Caki-2 and 786-O, respectively) and duplicate injections were made for each sample. Data processing was accomplished using two software tools: Scaffold (version Scaffold 3.0, Proteome Software Inc., Portland, OR) and Progenesis LC-MS (version 3.0, Nonlinear Dynamics, Durham, NC). Scaffold was employed to validate MS/MS-based peptide and protein identifications from Mascot database searching. Initial peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm [20]. Protein probabilities were assigned by the Protein Prophet algorithm [21]. Protein identifications were accepted, if they reached greater than 99% probability and contained at least 2 identified unique peptides. These identification criteria typically established < 0.01% false discovery rate based on a decoy database search strategy at the protein level. Extracted peptide intensities were evaluated with Progenesis LC-MS to obtain label-free relative quantification from the raw data files. The software created a single aggregate run based on LC retention time of each analysis to contain all MS data with distinguished peptide ions and, then, further feature outline maps were generated for detection and quantification of peptide ions from individual analyses using default program parameters. Peptide quantifications were performed by summing peak intensities followed by normalization, data filtering to retain only ions with positive charges (z) of two and three, and exporting peak lists to query against the IPI human protein sequence database using Mascot (see Database Search subsection above). Proteins were accepted as differentially expressed following analysis of variance (ANOVA, P<0.05), requiring at least a twofold change in expression and also passing validation by Peptide Prophet and Protein Prophet using protein identifications from Scaffold.
2013-08-12 | PXD000335 | Pride
Project description:Background: Although human gingival fibroblasts (hGF) and human periodontal ligament fibroblasts (hPDLF) exhibit numerous phenotypic similarities, it has been suggested that the secretory and behavioral differences, which exist between these cell types, are a result of the membrane protein composition of these cells. Methods: Four matched pairs of hGF and hPDLF were cultured. Prior to confluence, membrane bound and associated proteins from cells of the 4th passage were extracted. The processed protein samples were identified by digestion with trypsin and sequenced using capillary-liquid chromatography tandem mass spectrometry on an Thermo Scientific LTQ-Orbitrap XL mass spectrometer. Scaffold by Proteome Software was used to quantitate and validate protein identifications derived from MS/MS sequencing results. Results: Four hundred fifty proteins were common to both hGF and hPDLF. Of the proteins identified, 214 were known membrane bound or associated proteins and 165 proteins were known nuclear associated proteins. Twenty-seven proteins, identified from the 450 proteins, common to both hGF and hPDLF, were detected in statistically significant greater quantities in either hGF or hPDLF. More specifically, 13 proteins were detected in significantly greater quantities in hGF, while 14 proteins were detected in significantly greater quantities in hPDLF. Conclusions: Distinct differences in the cellular protein catalog may reflect the dynamic role and high energy requirements of hGF in extracellular matrix remodeling and response to inflammatory challenge as well as the role of hPDGF in monitoring mechanical stress and maintaining tissue homeostasis during regeneration and remineralization. Method Details: Sequence information from the MS/MS data was processed by converting the .raw files into a merged file (.mgf) using an in-house program, RAW2MZXML_n_MGF_batch (merge.pl, a Perl script). Isotope distributions for the precursor ions of the MS/MS spectra were deconvoluted to obtain the charge states and monoisotopic m/z values of the precursor ions during the data conversion. The resulting mgf files were searched using Mascot Daemon by Matrix Science version 2.3.2 (Boston, MA) and the database searched against the full SwissProt database version 2012_06 (536,489 sequences; 190,389,898 residues) or NCBI database version 20120515 (18,099,548 sequences; 6,208,559,787 residues The mass accuracy of the precursor ions were set to 20ppm, accidental pick of 13C peaks was also included into the search. The fragment mass tolerance was set to 0.5 Da. Considered variable modifications were oxidation (Met), deamidation (N and Q) and carbamidomethylation (Cys). Four missed cleavages for the enzyme were permitted. A decoy database was also searched to determine the false discovery rate (FDR) and peptides were filtered according to the FDR. The significance threshold was set at p less than 0.05 and bold red peptides is required for valid peptide identification. Proteins with a Mascot score of 50 or higher with a minimum of two unique peptides from one protein having a -b or -y ion sequence tag of five residues or better were accepted. Any modifications or low score peptide/protein identifications were manually checked for validation. Spectral Counting: Label Free Quantitation was performed using the spectral counting approach. Scaffold (version Scaffold_3.4.9, 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 (Keller, A et al Anal. Chem. 2002;74(20):5383-92). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 1 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii, AI Anal Chem. 2003 Sep 1;75(17):4646-58). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
2013-11-18 | PXD000387 | Pride
Project description:Proteomic response of adult Drosophila melanogaster acclimated at three contrasted thermal conditions (11, 25 and 31 degree Celsius) investigated by 2D-DIGE experiment. Thermal acclimation drastically alters thermotolerance of ectotherms, but the mechanisms determining this plastic response are not fully understood. The present study investigates the proteomic response (2D-DIGE) of adult Drosophila melanogaster acclimated at 11, 25 or 31 °C. As expected 11 °C-acclimation improved cold tolerance and 31 °C-acclimation improved heat tolerance. We hypothesized that the marked organismal responses to acclimation could be detected at the proteomic level assuming that changes in the abundance of specific proteins are linked to the physiological changes underlying the phenotypic response. The 31 °C-acclimated flies displayed a particular divergent proteomic profile where molecular chaperones made up a large number of the proteins that were modulated during heat acclimation. Many other proteins showed significant modulation during acclimation including proteins involved in iron ion and cell redox homeostasis, carbohydrate and energy metabolism, chromatin remodeling and translation, and contractile machinery. Interestingly the changes in protein abundance were often unrelated to transcriptional activity of the genes coding for the proteins, except for the most strongly expressed proteins (e.g. Hsp70). The 11 °C-acclimation evoked weak proteomic response despite the marked effect on the organismal phenotype. Thus the acquired cold tolerance observed here may involve regulatory process such as posttranslational regulation rather than de novo protein synthesis. Bioinformatics and data processing: The proteinScape 2.1 software (BrukerDaltonik GmbH) was used to submit MS/MS data to the following database: NCBI restricted to Drosophila (June 2011, 223,543 sequences) using the Mascot search engine (Mascot server v2.2, http://www.matrixscience.com). Parameters were set as follows: trypsin as enzyme with one allowed miscleavage, carbamidomethylation of cysteins as fixed modification and methionine oxidation as variable modifications. The mass tolerance for parent and fragment ions was set to 0.5 Da. Peptide identifications were accepted if the individual ion Mascot scores were above the identity threshold (the ion score is −10*log(P), where P is the probability that the observed match is a random event, P < 0.05). In case of ambiguous assignments (one compound fit to more than one peptide), peptide were accepted based on the peptide score, meaning that the peptide sequence with the highest score is accepted. The compilation of identified peptides to proteins was performed with the ProteinExtractor algorithm (Thiele et al., 2008, 2010), so that every protein reported was identified by at least one peptide with significant ion Mascot score (above the identity threshold).
2013-03-04 | PXD000043 | Pride
Project description: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.
2014-01-23 | PXD000647 | Pride
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.
2012-10-30 | PXD000056 | Pride
Project description:Saliva was collected from c.40,000 diet reared aphids, pooled and concentrated. Samples were coming from 2 aphid species: Metopolophium dirhodum and Sitobion avenae. Proteins were fractionated using SDS PAGE and visible bands were subjected to in gel digestion. Tryptic peptides were analysised on a Thermo LTQ-FT. Protein identification from the MS/MS data was performed using the TurboSEQUEST algorithm in BioWorks v. 3.2 (Thermo Fisher Scientific) to correlate the data against ACYPIproteins v2.1, the official protein set of the pea aphid genome assembly (33291 predicted protein models; accessed November 2011) available at http://www.aphidbase.com/aphidbase/downloads. The following search parameters were used: precursor-ion mass tolerance of 1.5 Da, fragment ion tolerance of 1.0 Da with methionine oxidation and cysteine carboxyamidomethylation specified as differential modifications and a maximum of two missed cleavage sites allowed. Two filters were applied: XCorr vs. charge state (1, 2, 3 and 4 = 1.50, 2.00, 2.50 and 3.00 respectively) and peptide probability (p<0.001). Matches with multiple unique peptides and a cumulative XCorr >20 are reported. PLoS One accepted: Proteomic profiling of cereal aphid saliva reveals ubiquitous and adaptative secreted proteins. Rao S.A.K., Carolan, J.C. and Wilkinson, T.L.W.
2013-04-09 | PXD000113 | Pride
Project description:In order to assess the quality of alleged PM identifications from Arabidopsis, PM-enriched fractions were compared to PM-depleted fractions using 18O isotopic labeling and mass spectrometry. The two samples submitted are biological replicates. Keywords: Protein Localization via MS Two biological replicates were analyzed in order to assess sample complexity. PM-derived vesicles were digested in 18O water and compared to PM-depleted fractions digested in 16O (natural abundance) water. Relative ion intensities were then used to calculate a heavy/lite ratio with proteins showing enrichment in the upper phase having a ratio greater than 1 or 0 on a log2 scale.
2005-06-21 | E-GEOD-2830 | biostudies-arrayexpress