The raw data were converted into a peak list format (.mgf) using the Mascot server (Matrix Science Ltd.). The resulting peaks were searched against the predicted protein sequences from the ERAC metagenome using the Mascot server (Matrix Science). The following search criteria were applied: carbamidomethylation as fixed modifications, oxidation of methionine as a variable modification, one missed trypsin cleavage, and a tolerance of 10 ppm for precursor ions and 1 Da for fragment ions. ScaffoldQ+ software was applied to further analyze the data processed by the Mascot server to validate the MS/MS-based peptide and protein identification. The following parameters were applied: a minimum protein probability of 90%, a minimum peptide probability of 50%, and a unique different minimum peptide of 2. The false-discovery rate (FDR) was adjusted to 1%. Protein quantification was based on the normalized spectrum abundance, which was calculated as the number of spectral counts identifying a protein. The presence of signal peptides and subcellular localization were manually assessed using the signal peptide prediction program SignalP (v.4.0) (89 (link)) and the TMHMM (v.2.0) server (90 (link)), respectively.
Mascot server
Manufactured by Matrix Science
Sourced in United Kingdom, United States, Germany, Japan
The Mascot Server is a software application that provides protein identification and characterization services. It is designed to analyze mass spectrometry data and match it against protein sequence databases to identify the proteins present in a sample. The Mascot Server operates as a centralized system, allowing multiple users to access and utilize the software's capabilities simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Proteome discoverer, by Thermo Fisher Scientific (26 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (5 mentions)
Proteinscape platform, by Bruker (4 mentions)
Proteome discoverer platform, by Thermo Fisher Scientific (4 mentions)
Autoflex speed, by Bruker (4 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (4 mentions)
Q exactive, by Thermo Fisher Scientific (3 mentions)
Amazon speed etd, by Bruker (3 mentions)
Ltq mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Mascot daemon, by Matrix Science (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Proteome discover, by Thermo Fisher Scientific (3 mentions)
Flexanalysis software, by Bruker (3 mentions)
3xflag peptide, by Merck Group (3 mentions)
Dataanalysis 4, by Bruker (3 mentions)
Nanoacquity hplc system, by Waters Corporation (2 mentions)
Gelcode blue staining, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (2 mentions)
Proteinlynx global server, by Waters Corporation (2 mentions)
173 protocols using mascot server
1
Metagenomic Protein Identification and Quantification
2
Quantitative Yeast Proteomics Analysis
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For peptide analysis, raw data processing and database searches were performed with Proteome Discoverer software 2.1.1.21 (Thermo Fisher Scientific). Peptide identifications were done with an in-house Mascot server version 2.5.1 (Matrix Science Ltd). MS2 data were searched against Saccharomyces cerevisiae sequences in SwissProt (release 2017_10). Precursor ion m/z tolerance was 8 ppm, and fragment ion tolerance was 0.5 Da. Tryptic peptides with up to two missed cleavages were searched. Propionamide on cysteines was set as a static modification. Oxidation of methionine, labels 13C615N2, and 2H4 on lysine were allowed as dynamic modifications. Mascot results were assigned q-values by the Percolator algorithm (45 (link)) version 2.05 as implemented in Proteome Discoverer. Spectra with identifications below 1% q-value were sent to a second round of database search with semitryptic enzyme specificity (one missed cleavage allowed) and 10 ppm MS1 mass tolerance (propionamide dynamic on Cys). In copurification experiments, only proteins were included if at least two peptides were identified with <1% false discovery rate. Typical false discovery rate values were ≤1% (peptide spectrum matches), 1.2% (peptides), and <1% (proteins). Only unique peptides were included in protein quantification.
3
Protein Identification by Mass Spectrometry
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To identify the proteins on the SDS-PAGE gels, bands were cut out from the gels and transferred to a microcentrifuge tube. Subsequently, in-gel reduction, alkylation, and tryptic digestion were carried out as previously reported by Miralles et al. [17 (link)]. An Autoflex speed MALDI-TOF/TOF (Bruker Daltonics, Bremen, Germany) instrument was used for mass spectra generation. The Mascot server (www.matrixscience.com , Matrix Science, London, UK) was used to carry out protein identification searches against a homemade database of Bertholletia excelsa and Pisum sativum proteins selected from the UniProt database (https://www.uniprot.org/ (accessed on 22 May 2020)).
4
Proteomic Identification and Quantification
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Raw data files were peak processed with Proteome Discoverer (version 2.1, Thermo Scientific) followed by identification using Mascot Server (version 2.2, Matrix Science, Boston, MA) against the Human (Swissprot) FASTA file (downloaded 12/2017). Search parameters included Trypsin/P specificity, up to two missed cleavages, a fixed modification of carbamidomethyl cysteine, and variable modifications of oxidized methionine, pyroglutamic acid for Q, and N-terminal acetylation. Assignments were made using a 10 ppm mass tolerance for the precursor and 0.05 Da mass tolerance for the fragments. All non-filtered search results were processed by Scaffold (version 4.8.7, Proteome Software, Portland, OR) utilizing the Trans-Proteomic Pipeline (Institute for Systems Biology) with threshold values set at 95% for peptides (0.2% false-discovery rate) and 99% for proteins (two peptide minimum), and quantitative comparisons were made using the label-free intensity-based absolute quantification (iBAQ) method with all samples normalized by total ion current for the run.
5
Proteomic Analysis of Glutathione Sepharose 4B Purification
6
Immunoprecipitation and Mass Spectrometry
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Immunoprecipitation was set up with 20 mg membrane extract and 50 µg of syngeneic or allogeneic IgG coupled to protein G magnetic beads and incubated for 16 hr at 4°C. Beads were washed thrice with MEB and bound protein complexes were eluted with 2X Laemmli buffer. The eluted sample was subjected to SDS-PAGE on a 4–12% Bis-Tris gel followed by GelCode Blue staining (Thermo Scientific) to visualize protein bands. Protein bands were excised, digested with trypsin and analyzed (MS Bioworks) using a nano LC/MS/MS with a NanoAcquity HPLC system (Waters) interfaced to a Q Exactive (Thermo Fisher). The mass spectrometer was operated in data-dependent mode, with MS and MS/MS performed in the Orbitrap at 70,000 FWHM and 17,500 FWHM resolution, respectively. The fifteen most abundant ions were selected for MS/MS. The data were processed with the Mascot Server (Matrix Science). Mascot DAT files were parsed into the Scaffold software for validation, filtering and to create a non-redundant list per sample. Data were filtered at 1% protein and peptide FDR, requiring at least two unique peptides per protein. Mass spectrometry analysis of precipitated proteins was performed once.
7
Immunoprecipitation and Mass Spectrometry
8
Protein Identification via Mass Spectrometry
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For protein identification, the MS/MS data were interpreted using a local Mascot server with MASCOT 2.6.2 algorithm (Matrix Science, London, UK) against an in-house database containing all Mus musculus and Rattus norvegicus entries from UniProtKB/SwissProt (version 2019_10, 50,313 sequences) and the corresponding 50,313 reverse entries. Spectra were searched with a mass tolerance of 10 ppm for MS and 0.07 Da for MS/MS data, allowing a maximum of one trypsin missed cleavage. Trypsin was specified as an enzyme. Acetylation of protein n-termini, carbamidomethylation of cysteine residues, and oxidation of methionine residues were specified as variable modifications. Identification results were imported into Proline software version 2.1 (http://proline.profiproteomics.fr/ ) for validation. Peptide Spectrum Matches (PSM) with pretty rank equal to one were retained. False Discovery Rate was then optimized to be below 1% at PSM level using Mascot Adjusted E-value and below 1% at Protein Level using Mascot Mudpit score.
Mass spectrometry data are available via ProteomeXchange [46 (link)] with the identifier PXD03002, associated with the doi: 10.6019/PXD030002.
Mass spectrometry data are available via ProteomeXchange [46 (link)] with the identifier PXD03002, associated with the doi: 10.6019/PXD030002.
9
Purification and MS Analysis of PSII-I Complexes
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PSII-I complexes were purified and desalted using Isolute C18 SPE cartridges (Biotage, Sweden). The columns were first washed and equilibrated, the sample diluted in 0,1% trifluoroacetic acid (TFA) and loaded onto the column. After washing with 2 ml 0.1% TFA, the proteins were eluted with 500 µl 80% acetonitrile (ACN), 20% water. The organic fraction was lyophilized in a vacuum concentrator (Eppendorf, Germany), reconstituted in 0.1% TFA and mixed in a 1:1 ratio with HCCA matrix solution (HCCA (alpha-cyano-4-hydroxycinnamic acid) saturated in 50% ACN, 50% water and supplemented with 0.1% TFA). Subsequently, 1 µl aliquots of the mixture were deposited on a ground steel MALDI target and allowed to dry and crystallize at ambient conditions. MS and MS/MS spectra were acquired on a prototype rapifleX MALDI-TOF/TOF (Bruker Daltonics, Germany) in positive ion mode. The Compass 2.0 (Bruker Daltonics, Germany) software suite was used for spectra acquisition and processing (baseline subtraction, smoothing, peak picking), a local Mascot server (version 2.3, Matrixscience, UK) was used for database searches against the T. elongatus proteome (UniProt, retrieved 4/2019) and BioTools 3.2 (Bruker Daltonics) was used for manual spectrum interpretation, de novo sequencing and peak annotation.
10
Immunoprecipitation and Mass Spectrometry Identification
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Immunoprecipitation was performed using PureProteome Protein G Magnetic Beads (Merck Millipore, Darmstadt, Germany) and the capture antibody, goat anti-rat IgG Fc (ab97086, Abcam), according to the manufacturer’s instructions. The bound immune complexes were analyzed by SDS-PAGE under reducing conditions. Protein bands were visualized by silver staining. Individual stained bands were excised from gels, destained, and subjected to enzyme digestion as described previously25 (link). The peptides were separated using a trypsin/lysyl endopeptidase solution (150 ng/30 μL), and amino acid sequences were determined by mass spectrometry (Easy-nLC 1000/Orbitrap Elite, Thermo Fisher Scientific). Mass spectrometry data were processed and subjected to database searches using the Mascot server (Matrix Science, Boston, MA, USA) or Sequest HT (Thermo Fisher Scientific).
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