Computational protocol: The Grapevine VvPMEI1 Gene Encodes a Novel Functional Pectin Methylesterase Inhibitor Associated to Grape Berry Development

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Protocol publication

[…] MS/MS analysis of recombinant VvPMEI1 was performed on protein after SDS-PAGE separation.The band excised from Coomassie stained gels was in-gel digested with trypsin (Promega) according to the procedure described by Shevchenko []. After enzymatic digestion, peptides were concentrated using ZipTip C18 reverse phase micro-columns (Millipore, Bedford, MA, USA) and analyzed by LC-MS/MS. The peptides were eluted over 180 min at 300 nl/min. using a 0–60% acetonitrile gradient in 0.1% formic acid using an Ultimate 3000 nano-chromatography pump (Thermo-Fisher Scientific). The peptides were eluted into a LTQ Orbitrap Discovery mass spectrometer (Thermo-Fisher, Bremen, Germany) operated in a data dependent mode. MS was acquired at 30.000 FWHM resolution in the FTMS (using a target value of 5 x105 ions) and MS/MS was carried out in the linear ion trap. Five MS/MS scans were obtained per MS cycle. Spectra were analyzed using Sequest (Thermo Fisher Scientific, San Jose, CA, USA; version 1.3.0.339). Sequest was set up to search the Vitis vinifera proteome database (Uniprot.org) assuming trypsin as the enzyme with 2 missed cleavage allowed. Sequest was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 ppm. Fixed modification of carbamidomethyl cysteine and variable modifications of oxidized methionine were considered in the search.For the proteomic analysis tissues were ground using a mortar and pestle and incubated at 4°C for 3 hours in a buffer containing 20mM sodium acetate, 1M sodium chloride and 1:100 v/v protease inhibitor (P9599; Sigma), pH 5.5. After centrifugation at 15000xg for 15 min at 4°C the supernatant was collected and protein concentration determined. Protein extracts were digested in solution with trypsin (Promega); the peptides were concentrated using StageTip C18 reverse phase micro-columns (Millipore, Bedford, MA, USA) and analyzed by LC-MS/MS. Peptides were eluted over 180 min at 300 nl/min. using a 0–60% acetonitrile gradient in 0.1% formic acid. Mass spectra were analyzed using the MaxQuant Software package. Raw data files were searched against the NCBInr proteome database Viridiplantae (containing 1,878,311 sequence entries) assuming trypsin as the enzyme with 2 missed cleavage allowed. Maxquant was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10.0 ppm. Fixed modification of carbamidomethyl cysteine and variable modification of oxidized methionine were considered in the search. MaxQuant identifications required FDR<0.01. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped. […]

Pipeline specifications

Software tools Comet, MaxQuant
Application MS-based untargeted proteomics
Organisms Vitis vinifera
Diseases Unverricht-Lundborg Syndrome