Computational protocol: Proteomics Analysis Reveals Novel RASSF2 Interaction Partners

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Protocol publication

[…] The RAW files were processed as an Electrospray DDA experiment in ProteinLynx Global ServerTM 3.0.2 (Waters, Milford, MA, USA). Both the electrospray survey and MSMS scans were calibrated to the 556.2771 Da/e leucine enkephalin lock mass (two lock spray scans with a lock mass tolerance of 0.1 Da). Adaptive noise reduction was used, and the deisotoped, processed spectra were exported as an mgf file for loading into Proteome Discoverer v1.4.1.114 (Thermo, Rockford, IL, USA) for analysis. The database used in Mascot v2.5.1 and SequestHT searches was the 10/29/2015 version of the UniprotKB Homo sapiens reference proteome canonical and isoform sequences. The database was annotated with the FASTA sequences pmKATE2-n vector, RASSF2 with HA tag, K-Ras with pmKATE2-n tag, and the nonhuman sequences from the 1/1/2012 version of the cRAP database were appended to it. Mass tolerances of 100 ppm for precursors and 0.1 Da for fragments were used in both search algorithms. In order to initiate a reverse database search, a Target Decoy PSM Validator node was included in the workflow. The resulting msf files from Proteome Discoverer were loaded into Scaffold Q+S v4.4.5 (Proteome Software, Portland, OR, USA). The false discovery rate for peptides was calculated using the Scaffold Local FDR algorithm. Protein probabilities were calculated using the Protein Prophet algorithm. Proteins were grouped by Scaffold protein cluster analysis to satisfy the parsimony principle. […]

Pipeline specifications

Software tools Proteome Discoverer, Scaffold Q+S
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Neoplasms