Whole cell shotgun analysis of substrate-adapted cells was performed as described recently [44 (link)]. Essentially, tryptic peptides were separated by a nanoLC system (UltiMate3000 nanoRSLC; ThermoFisher Scientific) operated in a trap-column mode and equipped with a 25 cm separation column (C18, 2 μm bead size, 75 μm inner diameter; ThermoFisher Scientific), applying a 280 min linear acetonitrile gradient. The nanoLC eluent was continuously analyzed by an online-coupled ion-trap mass spectrometer (amaZon speed ETD; Bruker Daltonik GmbH, Bremen, Germany) using a captive spray ion source (Bruker Daltonik GmbH). Per full scan MS (mass range 400–1400 m/z), 20 MS/MS spectra of the most intense doubly (or more highly) charged ions were acquired applying subsequent precursor exclusion for 0.2 min. Protein identification was performed using the ProteinScape platform (version 3.1; Bruker Daltonik GmbH) on an in-house Mascot server (version 2.3; Matrix Science Ltd, London, UK) based on the genome sequence of "A. aromaticum" EbN1 [6 (link)] and applying a target-decoy strategy as described [44 (link)]. Search results of the three biological replicates per test state were compiled and only proteins identified by at least 2 peptides were considered.
Proteinscape platform
Manufactured by Bruker
Sourced in United Kingdom
The ProteinScape platform is a comprehensive software solution for the management and analysis of mass spectrometry-based proteomics data. It provides a centralized environment for the storage, processing, and interpretation of proteomics data.
Automatically generated - may contain errors
Lab products found in correlation
Mascot server, by Matrix Science (4 mentions)
Amazon speed etd, by Bruker (3 mentions)
Ultimate 3000 nano rslc, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (1 mentions)
Captivespray ion source, by Bruker (1 mentions)
Mascot search engine, by Matrix Science (1 mentions)
Nanorslc system, by Thermo Fisher Scientific (1 mentions)
Amazon etd, by Bruker (1 mentions)
6 protocols using proteinscape platform
1
Quantitative Proteomics of Substrate-Adapted Cells
2
Proteomic Analysis of Rhodopsin Proteins
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Proteomic analysis was conducted using the rhodopsin-containing 25-kDa protein bands resolved by SDS-PAGE. These were excised from Coomassie-stained gels, washed, and tryptically digested as described by Wöhlbrand et al. (2016 (link)). Generated peptides were separated via nanoLC (UltiMate 3000 RSLCnano; Thermo Fisher Scientific, Germering, Germany) and analyzed by online coupling to electrospray ionization (CaptiveSpray ion source; Bruker Daltonik GmbH, Bremen, Germany) and mass analysis by a 3D ion trap (amaZon speed ETD; Bruker Daltonik GmbH) operated as described (Wöhlbrand et al. 2016 (link)). Protein identification was performed via the ProteinScape platform (version 3.1, Bruker Daltonik GmbH) on a Mascot server (version 2.3; Matrix Science, London, UK) against available rhodopsin sequences of O. marina also including translated EST data (obtained from NCBI, February 2016) and a target-decoy strategy applying described parameters (Wöhlbrand et al. 2016 (link)).
3
Proteomic Analysis of Arthritis Samples
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Acquired data were analysed against human protein sequences from Uniprot (downloaded 2015.01.17) using Mascot search engine (Matrix Science) incorporated into the ProteinScape™ platform (Bruker). In Mascot searches, tolerance of 10 ppm for precursor masses and 0.2 Da for fragment ions were specified. Searches were performed with trypsin specificity, two missed cleavages were allowed, and carbamidomethylation was set as static modification and oxidised methionine as dynamic modification. Identified proteins were filtered to a 1% FDR cut-off.
Normalised spectral abundance factor (NSAF) was calculated on the basis of peptide spectral counts for each protein detected in more than two arthritis samples. NSAF values were used in further analyses.
Normalised spectral abundance factor (NSAF) was calculated on the basis of peptide spectral counts for each protein detected in more than two arthritis samples. NSAF values were used in further analyses.
4
Peptide Separation and Identification
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Tryptic peptides were separated by nanoLC (UltiMate 3000 nanoRSLC; Thermo Fisher Scientific) applying the following setup: trap column (C 18 , 2 cm × 100 μm, 5-µm bead size; Thermo Fisher Scientific), separation column (C 18 , 25 cm × 75 µm, 2-µm bead size; Thermo Fisher Scientific), and a 280-min (shotgun) or 90-min (membrane, per gel fraction) linear gradient of acetonitrile (0-80% vol/vol). The eluent was continuously analyzed by an onlinecoupled ion trap mass spectrometer (amaZon speed ETD; Bruker Daltonics GmbH, Bremen, Germany) using settings as described by Zech et al. [92] . Per substrate conditions, six replicates (3 times each soluble and membrane protein-enriched fraction) were measured, giving rise to a total of 84 analyzed samples.
For protein identification, the ProteinScape platform (version 3.1; Bruker Daltonik GmbH), an in-house Mascot server (version 2.3; Matrix Science Ltd., London, UK), and the translated genome of P. vulgatus [93] were used. A target decoy strategy with described settings [94] was applied.
For protein identification, the ProteinScape platform (version 3.1; Bruker Daltonik GmbH), an in-house Mascot server (version 2.3; Matrix Science Ltd., London, UK), and the translated genome of P. vulgatus [93] were used. A target decoy strategy with described settings [94] was applied.
5
Shotgun Proteome Analysis of 4-Hydroxyacetophenone-Adapted Cells
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Cultures (400 ml each) of 4-hydroxyacetophenone-adapted cells of mutant strains as well as wild type 'A. aromaticum' EbN1 were harvested as described [Champion et al., 1999] to obtain cell material for whole-cell shotgun proteome analysis. Cell disruption, reduction, alkylation and tryptic digest were performed as described recently [Zech et al., 2013] . Tryptic peptides were analyzed using a nanoRSLC system (Thermo Fisher Scientific, Germering, Germany) in a trap column mode and equipped with a 25-cm analytical column (C18, 2 μm bead size; Thermo Fisher Scientific) coupled online to an ion-trap mass spectrometer (amazon ETD; Bruker Daltonik GmbH, Bremen, Germany). Protein identification was performed using the ProteinScape platform (version 3.1; Bruker Daltonik GmbH) on an in-house Mascot server (version 2.3; Matrix Science Ltd., London, UK) based on the genome sequence of 'A. aromaticum' EbN1 [Rabus et al., 2005] applying a target-decoy strategy as described by Zech et al. [2013] .
6
Secretome Proteomic Profiling Comparison
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The protein content from both the AP and MA secretome (100 μg) was precipitated with 100% acetone, overnight at -20 °C. Pellets were collected by centrifugation and resuspended in 6 M urea for tryptic insolution digestion, prior mass spectrometry (MS) analysis. Samples from 7 different individuals were analyzed per condition (AP and MA secretome). Protein identification and further comparison was performed with the Protein Scape platform (Bruker). IPA software was applied to evaluate the functional roles of the identified proteins.
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