The peak lists were generated using BioWorks software (version 3.3.1 Thermo Fisher, San Jose, CA), and then converted into mgf (mascot generic file) files. DBond search of the mgf files was conducted against the RNase A protein sequence (Swiss-Prot Number P61823) and a reversed version of Escherichia coli sequences (strain K12, Swiss-Prot release 2011.08, 4,430 entries) using the following parameters: ±15 ppm for mass accuracy of precursor ions, ± 0.8 Da mass tolerances of fragment ions, [DKR].* and *.[D] cleavage rules for Asp-N/C and trypsin, missed cleavage sites of up to 2, and oxidation of Met and deamidation of Asn as variable modifications. Among all search results, identifications were obtained with a false discovery rate (FDR) of 1% using a target-decoy approach.33 (link) FDR is calculated as D / T, where T and D are the numbers of target and decoy hits above score threshold, respectively. For the performance comparison, a MassMatrix21 (link) search was also conducted using the same parameters, and the pp score was used for the threshold in the FDR calculation.
Bioworks software
Manufactured by Thermo Fisher Scientific
Bioworks software is a data processing and analysis tool developed by Thermo Fisher Scientific. It is designed to streamline the management and interpretation of data generated from various laboratory instruments and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitors cocktail, by Roche (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Ltq mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Trypsin gold, by Promega (1 mentions)
Linear ion trap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using bioworks software
1
Tandem Mass Spectrometry Peptide Identification
2
Protein Identification via Mass Spectrometry
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Mass spectra were searched against the human International Protein Index (IPI) database (ftp://ftp.ebi.ac.uk/pub/databases/IPI/last_release/old/HUMAN/ipi.HUMAN.v3.35.fasta.gz. ) (IPI human v3.35 fasta with 68,348 entries) using the Bioworks software (v3.3.1; Thermo Fisher Scientific, Inc.) based on the Sequest algorithm. The search parameters included the following: i) Precursor ion mass tolerance <10 ppm; ii) fragment ion mass tolerance <1 Da; and iii) digestion mode was unspecific. The corresponding reversed sequence database was used to generate score criteria that yielded an estimated false positive rate of 1% (precision of 0.99). To minimize false positives, all output results were combined together using a homemade software to generate score criteria: The cross-correlation scores (Xcorr) of matches were >2.81, 3.22 and 3.41 for charged state 2, 3 and 4 peptide ions, respectively. To obtain reliable protein identification, only peptides with a ΔCn score >0.1 were used, and the ranks of the primary scores were 1, and the posterior error probability (PEP) <0.001.
3
Identification of MeCP2 Interactors
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Nuclei were extracted from the whole brain of WT and Mecp2-Flag mice as previously described[62 (link)]. Purified nuclei were resuspended in lysis buffer containing 20mM Tris, 150mM NaCl, 1.5mM MgCl2, 1mM EDTA, 10% Glycerol, 0.2% NP-40 and 1X proteinase inhibitors cocktail (Roche) and sonicated using a Misonix 3000. After centrifuging at 20,000g for 20min at 4°C, supernatant was incubated with 50ul of Anti-Flag M2 Magnetic Beads (Sigma) overnight at 4°C. In the following day, beads were washed with lysis buffer for 6 times. Bound protein was eluted by competition with 100 mg/ml of Flag Peptide (Sigma F3290). Eluted proteins from 5 IPs per genotype were pooled together and precipitated by adding 8 volume of pre-chilled acetone. Pellet was resuspended in 100mM Ammonium Bicarbonate solution. After DTT and IOAA treatment, protein was digested into peptides using Trypsin Gold (Promega) and Proteinase Max (Promega) overnight at 37°C. Peptides were separated by a nano HPLC and analyzed by a Thermo LTQ mass spectrometer. MS/MS spectra data was analyzed using Bioworks software (Thermo). Only proteins identified in Flag IP eluate from Mecp2-Flag mice but not WT mice were considered to be potential MeCP2-interacting proteins.
4
Phosphorylation Sites Identification in IIP4
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To identify the phosphorylation sites in IIP4, we incubated the purified recombinant His-tagged IIP4 and GST-tagged ILA1 together as described previously (Ning et al., 2011) . The resultant products were separated on SDS-PAGE gel and then stained with Coomassie brilliant blue. The gel containing IIP4 was sliced. After digestion by trypsin, the samples were subjected to a Linear ion trap mass spectrometer (Thermo Finnigan) for mass spectrometry analysis. The phosphorylated peptides were analyzed using Bioworks software (Thermo). This analysis was conducted three times.
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