To identify mitochondrial proteins that interact with Mitofilin with mass spectrometry, mitochondrial proteins from normal wild-type mice were immune-precipitated using an anti-Mitofilin antibody. Proteins eluted from the Mitofilin pull-down were separated by SDS-PAGE prior to mass spectrometry analysis. Gels were run, and ~2 cm regions of interest in each lane were subdivided into slices that were individually reduced/alkylated and digested with trypsin. The obtained digests were analyzed by HPLC-electrospray ionization-tandem mass spectrometry on an Orbitrap Velos Pro (Thermo Scientific, Waltham, MA, USA). Mascot (Matrix Science, Boston, MA, USA) was used to search the UniProt_mouse database and a database of common contaminants. The resulting Mascot files for the gel slices in each lane were combined for subset searching of the identified proteins by X Tandem, cross-correlation with the Mascot results, and determination of protein and peptide identity probabilities by Scaffold (Proteome Software, Houston, TX, USA). Relative quantities were determined by spectral counting. The most important identified proteins are represented in the table.
Mascot
Manufactured by Proteome Software
Sourced in United States, United Kingdom
Mascot is a software tool for the identification of proteins from mass spectrometry data. It performs database searches to match experimental mass spectrometry data against theoretical data generated from protein sequence databases.
Automatically generated - may contain errors
Lab products found in correlation
Mascot, by Matrix Science (26 mentions)
Scaffold 4, by Proteome Software (5 mentions)
Orbitrap velos pro, by Thermo Fisher Scientific (3 mentions)
Scaffold 3, by Proteome Software (2 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Ultimate3000 nano rslc systems, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Promega (2 mentions)
Lobind tube, by Eppendorf (1 mentions)
Ltq orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometry, by Thermo Fisher Scientific (1 mentions)
Nanoacquity hplc system, by Thermo Fisher Scientific (1 mentions)
Scaffold software, by Proteome Software (1 mentions)
Proteome discoverer, by Matrix Science (1 mentions)
Mascot software, by Matrix Science (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Scaffold version scaffold 4, by Proteome Software (1 mentions)
Ziptip, by Merck Group (1 mentions)
Proteome discoverer, by Thermo Fisher Scientific (1 mentions)
Nanoacquity hplc, by Waters Corporation (1 mentions)
32 protocols using mascot
1
Identification of Mitofilin-Interacting Mitochondrial Proteins
2
Proteomic Identification and Analysis
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The 15 most abundant ions were selected for MS/MS. Data were searched using the Mascot search engine with the SwissProt Human (forward and reverse appended with common contaminant proteins) database set to carbamidomethyl (C) fixed modification. Variable modification parameters were set to oxidation (M, Acetyl [N-term], Pyro-Glu [N-term Q]) and deamidation (N, Q). The peptide mass tolerance was set to 10 ppm and the fragment mass tolerance was set to 0.02 Da. A maximum of two missed cleavages and at least two unique peptides were required for protein identification. The false discovery rate was calculated for each MS experiment and is reported in Supplementary Data (MS Experiments). The resulting mass spectra were searched against the SwissProt database using Mascot (SwissProt_Human; forward and reverse; appended for common contaminant proteins), and the resultant Mascot DAT files were parsed into Scaffold (Proteome Software, Portland, OR) for validation, filtering, and generation of nonredundant identifications. Gene Ontology (GO) analysis was conducted using GO Enrichment (geneontology.org). During the process of uploading protein identifications, proteins identified in Scaffold with ambiguous association to genes in the Ingenuity Pathway Analysis database were excluded from the analysis.
3
Proteomic Identification of NpdA
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Methods outlined by Shetty et al. [10 (link)] were followed for protein identification by mass spectrometry. Briefly, the putative NpdA band recovered in the SLP extract was excised from the SDS-PAGE gel, the gel slice chopped into small pieces, and the pieces transferred to a LoBind tube (Eppendorf; Enfield, CT) for further processing and Trypsin digestion. Tryptic digests were analyzed by LTQ Orbitrap XL (ThermoScientific; Waltham, MA). Mass spectral data was searched against the P. citrulli AAC00-1 genome by using Mascot (Matrix Science, London, UK; version Mascot) and peptide assignments made with Mascot were validated with Scaffold (Proteome Software Inc., Portland, OR).
4
Identification of Mitofilin Interactome
5
Comparative Proteomics of Young and Aged Rat Exosomes
6
Mass Spectrometry-based Protein Identification
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All MS/MS data were searched against the UniProt/Swiss-Prot (release 2012_03) database using Mascot (version 2.2.06, Matrix Science, London, UK), in which the peptide and fragment mass tolerances were 10 ppm and 0.8 Da, respectively, and up to two missed cleavages were allowed for errors in trypsin specificity. For variable peptide modifications, methionine oxidation and formylation of lysine, arginine, and N-terminal amino acids were taken into account. Reported results were obtained from triplicate LC-MS runs for each sample with all peptide hits included. Unique peptides and proteins were identified by the following proteomics guidelines. Mascot search results were processed through Scaffold software (version 3.3.3, Proteome Software, Portland, OR) for gene ontology analyses and validation of MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at a Scaffold peptide probability of >95%. Protein identifications were accepted if they could be established at a Scaffold protein probability of >99% and contained at least two identified peptides. Identified proteins were also analyzed in terms of putative functional association networks using the STRING 9.01 Server (http://www.string-db.org ).
7
Proteomic Analysis of Protein Modifications
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MS sequencing and database analyses was performed by the Johns Hopkins Mass Spectrometry and Proteomics Facility as previously described [75 (link), 76 ]. Peptide sequences were identified using Proteome Discoverer and Mascot software (Matrix Science) to search the NCBInr 167 database, including gly-gly modifiation on lysine as a variable protein modification. False discovery rate (FDR) was set at 1.0. Mascot search result *.dat files were processed in Scaffold (Proteome Software, Inc.) to validate protein and peptide identifications. Exclusion criteria for proteins are described in the Results section.
Functional analyses of gene lists obtained by MS sequencing utilized DAVID v6.7 (Database for Annotation, Visualization and Integrated Discovery [83 (link)]). Protein multiple alignments used Clustal Omega 1.2.1 (EMBL-EBI) and the ESPript 3.0 web sever (http://espript.ibcp.fr/ESPript/ESPript/ ). RNA-Seq datasets from public databases (GEO GSE67196 and NeuroLINCS dbGaP Study phs001231 SRP098831) were analyzed for gene expression using the software package TETranscripts as previously described [84 (link), 85 (link)]. Ubiquitination prediction algorithms included UbPred (http://www.ubpred.org , [86 (link)]), BDM-PUB (http://bdmpub.biocuckoo.org ), and UbiSite (http://csb.cse.yzu.edu.tw/UbiSite/ ).
Functional analyses of gene lists obtained by MS sequencing utilized DAVID v6.7 (Database for Annotation, Visualization and Integrated Discovery [83 (link)]). Protein multiple alignments used Clustal Omega 1.2.1 (EMBL-EBI) and the ESPript 3.0 web sever (
8
Detailed Protein Identification Workflow
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Peak lists were generated from raw orbitrap data using the EasyProtConv conversion tool (version 1.6) from the EasyProt software platform. The peak list files were searched against the SwissProt database (release 15.10 of 21-Sept-2011) using Mascot (version 2.2.0; Matrix Science, Ltd., London, UK). Human taxonomy (20,323 sequences) was specified for database searching. The parent ion tolerance was set to 10 ppm. Amino acid modifications were oxidized using methionine and carbamidomethyl cysteine. Trypsin was selected as the enzyme, with one potential missed cleavage, and the normal cleavage mode was used. The Mascot search was validated using Scaffold 3.6.5 (Proteome Software, Portland, OR, USA). Proteins matching two alternate peptides with a minimum probability score of 95% were selected for further analysis.
9
Co-Immunoprecipitation and Mass Spectrometry
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Whole cell extracts (WCE) were prepared and co-IP performed essentially as described previously (Johnson et al., 2001 (link)). Co-IPs used either 5 µg affinity-purified mouse monoclonals (MAbs), or 5–10 µg purified IgG fractions from rabbit polyclonal antisera, coupled to protein G- and/or protein A-sepharose beads (GE Healthcare UK Ltd.). Proteins were eluted from beads with 0.2 M glycine HCl pH2.5. Samples were neutralised by addition of 0.1 volume 1 M Tris HCl ph8.5. After elution, proteins were precipitated with chloroform and methanol and subjected to in-solution tryptic cleavage as described previously (Gloeckner et al., 2009 (link)). LC-MS/MS analysis was performed on Ultimate3000 nano RSLC systems (Thermo Scientific) coupled to a Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) by a nano spray ion source (Boldt et al., 2016 (link)). Mascot (Matrix Science, Version 2.5.1) was used to search the raw spectra against the human SwissProt database for identification of proteins. The Mascot results were verified by Scaffold (version Scaffold_4.8.8, Proteome Software Inc, Portland, OR, USA) to validate MS/MS-based peptide and protein identifications.
10
Comparative Proteomic Analysis of Bacterial Stress Responses
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Data were searched using a local copy of Mascot (Matrix Science) with the following parameters: enzyme, trypsin/P; database, https://www.ncbi.nlm.nih.gov/nuccore/CP000057.2 (concatenated forward and reverse plus common contaminants); fixed modifications, carbamidomethyl (C); variable modifications: acetyl (N-term), deamidation (N,Q), oxidation (M), pyro-glu (N-term Q); mass values, monoisotopic; peptide mass tolerance, 10 ppm; fragment mass tolerance, 0.02 Da; maximum missed cleavages, 2. Mascot DAT files were parsed into Scaffold (Proteome Software) for validation, filtering and to create a non-redundant list per sample. Data were filtered using a 1% protein and peptide false discovery rate (FDR), requiring at least two unique peptides per protein. We used the normalized spectral counts for downstream analysis. To evaluate the variation and reproducibility in the replicates, we generated the PCA plot with 95% confidence ellipses surrounding each population (using the FactoMineR and ggplot2 packages in R), with the normalized spectral counts for each protein identified by mass spectrometry in the three samples of planktonic, anti-rsPilA NRel, and anti-IHF NRel groups [60 (link),61 ].
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