The data were analyzed using the ProteinScape (Bruker Daltonics) database software, and MASCOT 2.3 (Matrix Science, London, UK) was used as a search engine. The search parameters were as follows: carbamidomethylation of cysteine residues; precursor-ion mass tolerance, +/−0.3 Da; and fragment-ion mass tolerance, +/−0.5 Da. For data from the iTRAQ-labeling procedures, the iTRAQ 4-plex (peptide label) modification was also used in the search. The false discovery rate (FDR) for peptide identification was 0.05 in all analyses. The SwissProt and NCBI databases were used for protein identification. To reduce false-positive protein identifications, only proteins identified in both databases were considered significant hits. The plasma proteome database, available for free at http://www.plasmaproteomedatabase.org , was used for functional annotation of all identified proteins [22] (link). All comparisons and compiled lists of identified proteins were generated using the ProteinScape software (Bruker Daltonics).
Proteinscape
Manufactured by Bruker
Sourced in Germany, United Kingdom
ProteinScape is a comprehensive software platform for the management and analysis of proteomics data. It provides a centralized database and workflow-based data processing capabilities to support various proteomics techniques and applications.
Automatically generated - may contain errors
Lab products found in correlation
Mascot, by Matrix Science (3 mentions)
Data analysis software, by Bruker (3 mentions)
Flexanalysis, by Bruker (2 mentions)
Nanoelute, by Bruker (2 mentions)
Trypsin, by Promega (2 mentions)
Mascot search engine, by Matrix Science (1 mentions)
Amazon etd, by Bruker (1 mentions)
Captivespray ion source, by Bruker (1 mentions)
Amazon speed etd ion trap mass spectrometer, by Bruker (1 mentions)
Ziptip, by Merck Group (1 mentions)
384 mtp anchorchip, by Bruker (1 mentions)
Trypsin gold, by Promega (1 mentions)
Autoflex speed, by Bruker (1 mentions)
Mascot server, by Matrix Science (1 mentions)
15 t solarix xr ft icr mass spectrometer, by Bruker (1 mentions)
1290 series uhplc system, by Agilent Technologies (1 mentions)
Mn1000, by Thermo Fisher Scientific (1 mentions)
Colloidal blue, by Thermo Fisher Scientific (1 mentions)
Ultraflextreme maldi mass spectrometer, by Bruker (1 mentions)
Anti tau antibody, by Agilent Technologies (1 mentions)
21 protocols using proteinscape
1
Plasma Proteome Identification and Analysis
2
Identifying PBF-interacting Proteins
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PBF-interacting proteins were isolated by coimmunoprecipitation (anti-mHA antibody) and separated by SDS-PAGE. Following destaining, proteins were reduced, alkylated, and trypsinized before undergoing HPLC and AmaZon ETD ion trap and tandem mass spectrometry (Bruker Daltronics). Mass spectrographs were analyzed via the Mascot search engine (Matrix Science) and ProteinScape (Bruker). Multiple runs were performed to identify consistent interactors.
3
Trypsin Digestion and Protein Identification
4
Mass Spectrometry-Based Protein Identification
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Purified protein complexes were precipitated by the addition of 20% trichloroacetic acid and resolved with 50 mm ammonium bicarbonate solution. To mask the thiol group, samples were treated with 10 mm DTT at 60 °C for 30 min and then reacted with 10 mm iodoacetamide. Protein digestion was performed by the addition of 1 U·mL−1 Trypsin Gold (Promega, Madison, WI, USA) overnight. Digested peptides were prepared by a treatment with de‐salting by Zip‐Tip (Merck Millipore, Burlington, MA, USA). Peptides were then separated by silica‐based reverse‐phase chromatography (gradient with acetonitrile: 0–60% in 0.1 m trifluoroacetic acid) and spotted on MTP AnchorChip 384 (Bruker Daltonics, Billerica, MA, USA) with mixing with α‐cyano‐4‐hydroxy cinnamic acid. Spotted peptides were analyzed by Autoflex speed (Bruker Daltonics), an analyzer for MALDI‐TOF/TOF with the protocol, Protein Scape (Bruker Daltonics), and proteins were identified by Mascot server (Matrix Science, Tokyo, Japan).
5
Purification and Identification of Protein Complexes
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A procyclic cell line expressing TAP–RBP33 from the endogenous locus was generated using the strategy described in (53 (link)). Protein complexes were purified from 1–2 × 1010 procyclic trypanosomes using the TAP method (54 (link)) with the modifications described in (53 (link)). TAP-purified material was subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) in EASY-nLC equipment (Proxeon) coupled to an amaZon speed ion ETD trap (Bruker). Proteins were identified using ProteinScape (Bruker) and MASCOT (Matrix Science) software. We considered only those proteins showing a score >60 and with two or more detected peptides.
6
Targeted Proteomics Using FT-ICR MS
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The CtFDOdegl sample was diluted in 0.5 M glycine buffer pH 2.3 and 300 pmol was injected onto a pepsin column for online protein digestion. After 3 min of digestion and trapping, the peptides were separated by a 1290 series UHPLC system (Agilent Technologies) on a reverse-phase C18 column linked to the electrospray ion source of a 15T SolariX XR FT-ICR mass spectrometer (Bruker Daltonics). The mass spectrometer was operated in positive data-dependent mode. Data were processed using the DataAnalysis 4.2 software and exported to mgf format. ProteinScape (Bruker Daltonics) with the Mascot search engine was used for the identification of peptides.
7
Tau Immunoprecipitation and Mass Spectrometry
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Tau was immunoprecipitated from 1 mg of total protein extracted from iPSC-derived neurons (120 DIV) using a polyclonal anti-tau antibody (Dako Cytomation). Immunoprecipitated samples were analyzed by western blot using a monoclonal tau antibody (MN1000; Thermo Fisher Scientific) or stained with colloidal blue (Thermo Fisher Scientific). Bands that corresponded to tau by western blot analysis were excised from the colloidal blue SDS-PAGE. Peptide masses of digested protein samples were determined using a Bruker ultrafleXtreme Maldi mass spectrometer in reflectron mode and ms/ms fragmentation performed in LIFT mode. Data analysis was with FlexAnalysis, BioTools and ProteinScape software (Bruker). Database searches of the combined mass fingerprint-ms/ms data were performed using Mascot (http://www.matrixscience.com ).
8
Proteomic Analysis of Rat Samples
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Data from mass spectrometry were processed using a Compass 1.7 for OTOF (Bruker Daltonics, Billerica, MA, USA) and deconvoluted to generate a file compatible with Mascot. This file was analyzed using Protein Scape (Bruker Daltonics, Billerica, MA, USA), a program that uses Mascot algorithms to perform the search. The database used was Swissprot, an annotated protein sequence database. The taxonomy was rattus, and the enzyme was trypsin with two missed cleavages. The fixed modification was carbamidomethylation, and the variable modifications were oxidation of methionine and acetylation of lysine and arginine. Mass tolerance modification was 150 ppm to 1 Da. Mascot analysis of all proteins (p < 0.05) used a minimum score of 35.
9
Flat-Mounted Deconstructed Membranes for Protein Imaging
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For protein imaging, flat-mounted DMs (Samples ID C7 and F7 shown in Table 1 ) were washed with 70 to 100% ethanol and then sprayed with 10 mg/mL a-cyano-4-hydroxycinnamic acid in 70% acetonitrile containing 1% TFA using an automated sprayer (TM-Sprayer; HTX technologies, Chapel Hill, NC). Mass spectra were measured using a Rapiflex Tissuetyper (Bruker) with a spatial resolution of 50 μL. The DMs were then sprayed with trypsin solution (25 mg/mL in 20 mM aqueous NH4HCO3, pH 7.5–8.5) at room temperature and incubated for 2 h at 50 °C. MALDI-IMS data were obtained and analyzed using flexImaging 5.0 and SCiLS Lab 2018b (Bruker). For protein analysis, PEAKS Studio 8.5 (Bioinformatics Solutions Inc., Ontario, Canada), ProteinScape (Bruker), and MASCOT software (Matrix Science, London, UK) were employed. Samples ID C3-6 and F3-6 (Table 1 ) were used for preliminary experiments to confirm that MALDI-IMS is feasible in flat-mounted DMs.
10
Proteomics Workflow for Human Samples
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The MS data was exported to ProteinScape (Version 3.1.0 348; Bruker Daltonics). The file for both separation methods was combined into one for each sample. Protein searches were conducted using Mascot Server v 2.5.1 (Matrix Science, UK). Spectra were searched against the Swissprot Homo sapiens database. Semitrypsin was selected as the enzyme specificity allowing up to two missed cleavages. The MS error tolerance was set to 0.3 Da and the MS/MS error tolerance was set to 0.8 Da. Peptide and protein Mascot threshold scores were set to 20 and 80, respectively. Instrument specificity was set to ESI-TRAP. The modifications included were carbamidomethyl (C) as fixed and oxidation (M), ammonia loss (N-term-C), sodium (DE) and deamidation (NQ) as variable modifications. Protein identifications required at least one unique peptide identification from the list of identified peptides for that protein.
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