Affinity purified YULINK was processed by in-gel digestion. AcGFP-YULINK protein was purified by using GFP-trap beads following the IP protocol described above, with three additional washes with wash buffer. A portion of the purified YULINK was separated on SDS-PAGE and peptides were recovered using the in-gel method. Peptides from in-gel samples were loaded onto C18 spin columns (Thermo Fisher Scientific, Rockford, lL, USA). Peptides were eluted from columns with 70% acetonitrile and separated on a C18 column using an online nano-LC (Proxeon, Odense, Denmark) coupled to an LTQ-orbitrap velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). In each full scan the 10 most abundant peptides were selected for higher-energy C-trap dissociation (HCD) fragmentation. Raw data files were processed and analyzed using Mascot Daemon software (Matrix Science, Boston, USA).
Mascot daemon software
Manufactured by Matrix Science
Sourced in United Kingdom, United States
Mascot Daemon software is a tool for processing mass spectrometry data. It provides a platform for automated analysis and identification of proteins within complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Hystar version 3, by Bruker (3 mentions)
Compass dataanalysis software version 3, by Bruker (3 mentions)
Microtof q 2, by Bruker (3 mentions)
Ultimate 3000 nano lc system, by Thermo Fisher Scientific (2 mentions)
Formic acid, by Merck Group (2 mentions)
C18 spin column, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Rna labeling mix, by Roche (1 mentions)
T3 t7 rna polymerase, by Roche (1 mentions)
Microtof q, by Bruker (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
C18 column, by Thermo Fisher Scientific (1 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
Triple tof 4600 mass spectrometer, by AB Sciex (1 mentions)
Mascot distiller, by Matrix Science (1 mentions)
2d clean up kit, by Bio-Rad (1 mentions)
Protein extraction kit, by Bio-Rad (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
18 protocols using mascot daemon software
1
Affinity Purification of YULINK Protein
2
Phosphoproteomics of Peptide Mixtures
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Peptide mixture was analysed by an Ultimate® 3000 Nano-LC systems (Thermo Scientific, USA) coupled with a microTOF-Q II (Bruker, Germany). The acquisition was controlled by HyStar™ version 3.2 (Bruker, Germany). MS and MS/MS spectra covered the mass range of m/z 400–2000 and m/z 50–1500, respectively. The raw data format (.d) files were processed and converted to mascot generics files (.mgf) using Compass DataAnalysis™ software version 3.4 (Bruker, Germany) and submitted for database searches using Mascot Daemon software (Matrix Science, USA) against in-house transcriptomics database. Miss cleavage was allowed at one. Variable modifications were set as carbamidomethyl (C), oxidation (M), phospho (ST), and phospho (Y), MS peptide tolerance was 0.8 Da and MS/MS tolerance was 0.8 Da. Differential phosphoproteins were classified by gene ontology using Blast2Go software. Protein domains were predicted by Pfam 32.0 (September 2018, 17929 entries). Swiss model server was used for three-dimensional (3D) structure modeling71 (link). The template was selected by a sequence with the highest percentage identity. The.pdb file of modeled protein structures were downloaded and analyzed by Visual Molecular Dynamics software72 .
3
Proteasome and Pragmin Interactor Identification
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Proteasome 20S. The proteasome dataset was retrieved from 33 (link) . Briefly, proteasome complexes were immuno-purified from in vivo formaldehyde crosslinked human U937 cells using the MCP21 antibody targeting the α-2 subunit of the 20S core particle. After trypsin digestion and extraction, peptides were identified by nano-liquid chromatography (LC) MS/MS. The database search was performed using the Mascot Daemon software (version 2.3.2, Matrix Science).
Detailed information can be found in the original paper 33 (link) .
Pragmin. The Pragmin dataset was retrieved from 34 (link) . Note that the 61 proteins of this dataset do not contain the Pragmin itself. Briefly, a SILAC-based quantitative proteomic analysis, which allowed the identification in a semi-quantitative manner of Pragmin interactors in FLAG-Pragmin transfected human HEK293T cells, was undertaken. It reproducibly yielded more than 52 specific interactors, with a mean ratio >4 and found two protein-kinases that were prominently associated with Pragmin (mean ratio >12), including the Ser/Thr kinases AMPK and the TK CSK. Previous Pragmin interactors such as Src 35 (link) and SgK269/PEAK1 36 were, however, not recovered in our SILAC analysis. Detailed information can be found in the original paper 34 (link) .
Detailed information can be found in the original paper 33 (link) .
Pragmin. The Pragmin dataset was retrieved from 34 (link) . Note that the 61 proteins of this dataset do not contain the Pragmin itself. Briefly, a SILAC-based quantitative proteomic analysis, which allowed the identification in a semi-quantitative manner of Pragmin interactors in FLAG-Pragmin transfected human HEK293T cells, was undertaken. It reproducibly yielded more than 52 specific interactors, with a mean ratio >4 and found two protein-kinases that were prominently associated with Pragmin (mean ratio >12), including the Ser/Thr kinases AMPK and the TK CSK. Previous Pragmin interactors such as Src 35 (link) and SgK269/PEAK1 36 were, however, not recovered in our SILAC analysis. Detailed information can be found in the original paper 34 (link) .
4
Arabidopsis Thaliana Proteome Profiling
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For Mass spectrometric analyses, samples were run in 1D SDS-PAGE gel and stained by silver nitrate [70 (link)]. The SDS-PAGE gels were cut into pieces according to the silver staining result. The gel pieces were digested with trypsin (Promega) overnight, and then analyzed via liquid chromatography combined with electrospray tandem mass spectrometry on an LTQ Orbitrap (Thermo Fisher) with lock mass calibration. All of the raw data files were searched against Mascot Daemon software (Version 2.3.0, Matrix Science, London, UK) based on the Mascot algorithm. The database used was Swiss-Prot (Taxonomy: Arabidopsis Thaliana; release 2012_12_28, with 11571 entries). To reduce false positive identification results, a decoy database containing the reverse sequences was appended. The searching parameters were set up as following: full trypsin (KR) cleavage with two missed cleavage sites was considered. Oxidation on methionine and acetylation of the protein N-terminus were set as variable modifications. The peptide mass tolerance was 20 ppm and the fragment ion tolerance was 1.0 Da. Peptides with Percolater scores exceeding 13 were accepted as correct matches, the FDR is 0.01 (refer to S1 Text for details).
5
Identifying linc1281-binding proteins in mESCs
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To identify linc1281-interacting proteins in mESCs, biotin-RNA pull down and subsequent liquid chromatography-mass spectrometry were performed. Briefly, biotin-labeled RNAs were synthesized by using RNA Labeling Mix (Roche, 11685597910) and T3/T7 RNA polymerase (Roche, 10881767001/11031171001). Linc1281 or antisense RNAs were heated to 90°C for 2 min, held on ice for 2 min and then incubated in room temperature for 20 min in RNA structure buffer (10 mM Tris–HCl pH 7.0, 0.1 M KCl and 10 mM MgCl2) to form proper secondary structure. mESCs (1 × 107 cells) were lysed with RIP lysis buffer and Immunoprecipitation was performed by using streptavidin beads coated with 3 μg of linc1281 or antisense RNAs. The RNA-binding proteins were sequenced and identified by LC–MS as described previously (41 (link)). The mass spectra were searched using the Mascot Daemon software (Version 2.3.0, Matrix Science, London, UK) based on the Mascot algorithm. The database used to search was the Mouse UniProtKB database.
6
Venom Peptide Identification via LC-MS/MS
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Venom peptides were dissolved in 0.1% formic acid (Sigma-Aldrich, USA) and subjected to an Ultimate® 3000 Nano-LC system analysis (Thermo Scientific, USA). The peptides were eluted and infused with a microTOF-Q II (Bruker, Bremen, Germany). The acquisition was operated by HyStar™ version 3.2 (Bruker, Germany). The data were processed and converted to mascot generics files (.mgf) using Compass DataAnalysis™ software version 3.4 (Bruker, Germany). The database search was performed using Mascot Daemon software (Matrix Science, Boston, MA, USA) against Chordata NCBI database with the following parameters: one missed cleavage site, variable modifications of carbamidomethyl (C) and oxidation (M), 0.8 Da for MS peptide tolerance, and 0.8 Da for MS/MS tolerance. The significance threshold was set at 95%. Three biological replications were performed for protein identification. The obtained proteins were classified according to their gene ontology using Blast2Go software.
7
Quantitative Proteomic Analysis of Samples
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MS/MS data acquired with LC-MS/MS was performed using the Mascot Daemon software (version 2.4, Matrix Science, UK). Data were searched against human entries in the SwissProt (2012). Carbamidomethylation of cysteine was set as a static modification and oxidation of methionine was set as a variable modification. The mass tolerance was set to 20 ppm and 0.1 Da for precursor and fragment, respectively. The false discovery rate (FDR) by searching decoy databases was estimated to be 1.0%. The abundance of identified proteins was estimated by calculating emPAI, which is one of semi-quantitative approaches widely used in comparative proteomic studies21 (link)-23 (link). The emPAI is an exponential form of PAI (the number of detected peptides divided by the number of observable peptides per protein).
8
Protein Identification by Database Searching
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The Mascot Daemon software (version 2.3.0, Matrix Science, London, UK) was used to perform database searches against Homo sapiens entries in Uniprot protein database. The mass tolerances in MS and MS/MS were set to 5 ppm and 0.6 Da, respectively, and the instrument setting was specified as “ESI-Trap”. Mascot results were parsed with the in-house-developed software Mascot File parsing and Quantification (MFPaQ) v4.0.0 software (http://mfpaq.sourceforge.net/ ), and protein hits were automatically validated if they satisfied one of the following criteria: identification with at least one top-ranking peptide with a Mascot score of more than 39 (P-value <0.001) or at least two top-ranking peptides each with a Mascot score of more than 22 (P-value <0.05). When several proteins matched exactly the same set of peptides, only one member of the protein group was reported in the final list. To evaluate the false-positive rate in these experiments, all the initial database searches were performed using the “decoy” option of Mascot. Mascot results were parsed with the in-house-developed software MFPaQ v4.0.0 (http://mfpaq.sourceforge.net/ ), and protein hits were automatically validated.
9
Mascot-based proteomic database search
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The Mascot Daemon software (version 2.3.2, Matrix Science, London, UK) was used to perform database searches in batch mode with all the raw files acquired on each sample. Data were searched against all entries in the SwissProt Human Mouse_20090127 protein database (36322 sequences; 19974433 residues). Oxidation of methionine was set as a variable modification for all Mascot searches. Specificity of trypsin digestion was set for cleavage after Lys or Arg except before Pro, and one missed trypsin cleavage site was allowed. The mass tolerances in MS and MS/MS were set to 5 ppm and 0.6 Da, respectively. Mascot results were parsed with the in-house developed software Mascot File Parsing and Quantification (MFPaQ) version 4.0 and protein hits were automatically validated if they satisfied a false discovery rate (FDR) of 1% with peptides of a minimal length of 8 amino acids [36 (link)].
10
Quantitative Proteomic Analysis of Rat
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The Mascot Daemon software (version 2.4.0, Matrix Science, London, UK) was used to search the MS/MS data against the SwissProt_rat database (release 2013_07; taxonomy: Rattus; containing 9,354 sequences). The carbamidomethylation of cysteines was set as a fixed modification. The oxidation of methionine and protein N-terminal acetylation were set as variable modifications. The specificity of trypsin digestion was set for cleavage after K or R, and two missed trypsin cleavage sites were allowed. The mass tolerances in MS and MS/MS were set to 10 ppm and 0.5 Da, respectively. Peptide and protein identifications were validated using Scaffold (version 4.0.1, Proteome Software Inc., Portland, OR, USA). Peptide identifications were accepted if they were detected with ≥95.0% probability by the Scaffold local false discovery rate algorithm, and protein identifications were accepted if they were detected with ≥99.0% probability and contained at least 2 identified peptides (Nesvizhskii et al., 2003 (link)). Label-free quantification was performed using the Progenesis LC-MS software (version 4.1, Nonlinear, Newcastle upon Tyne, UK), as previously described (Stoop et al., 2013 (link)).
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