Computational protocol: An improved protocol to study the plant cell wall proteome

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Protocol publication

[…] Following spot detection, all visible spots of each of the 3 gels were stored in a picklist and picked with an Ettan Spotted Picker (GE Healthcare). Digestion and MALDI spotting were carried out using a Freedom EVO II workstation (Tecan). Briefly, gels plugs were washed for 20 min in a 50 mM ammonium bicarbonate solution in 50% v/v MeOH/MQ water and dehydrated for 20 min with 75% ACN. After dehydration, proteins were digested with trypsin Gold (Promega), 8 μl of a solution containing 5 ng/μL trypsin in 20 mM ammonium bicarbonate (overnight, 37°C). After digestion, peptides were extracted from the gel plugs with 50% v/v ACN containing 0.1% v/v TFA and dried. Peptides were then solubilized in 2 μL of 50% v/v ACN containing 0.1% v/v TFA and 0.7 μL was spotted on MALDI-TOF targets. A volume of 0.7 μL of 7 mg/mL α-cyano-4-hydroxycinnamic acid in 50% v/v ACN containing 0.1% v/v TFA was added. A MALDI peptide mass spectrum was acquired using the AB Sciex 5800 TOF/TOF (AB Sciex), and the 10 most abundant peaks, excluding known contaminants, were automatically selected and fragmented. MS analyses were carried out as described by Printz et al. (). MS and MS/MS spectra were submitted for NCBInr database-dependent identification using the taxonomy viridiplantae (http://www.ncbi.nlm.nih.gov) downloaded on September 23, 2013 and containing 32,770,904 sequences on an in-house MASCOT server (Matrix Science, www.matrixscience.com). A second search was carried out against an EST fabacea database downloaded on December 17, 2013 and containing 19,932,450 sequences. The parameters used for these searches were mass tolerance MS 100 ppm, mass tolerance MS/MS 0.75 Da, fixed modifications cysteine carbamidomethylation, and variable modifications methionine oxidation, double oxidation of tryptophan, and tryptophan to kynurenine. Proteins were considered as identified when at least two peptides passed the MASCOT-calculated 0.05 threshold scores (respectively a score of 50 for all NCBI viridiplantae queries and 57 for the EST fabacea queries). [...] A volume of 5 μL of the extracted peptides were desalted and separated by reverse phase separation using an Eksigent nano 1DLC (AB Sciex) coupled with a LTQ-OrbiTrap XL mass spectrometer (Thermo scientific) operated with Xcalibur (2.0.7 SP1). Peptide desalting was carried out on C18 OMIX tips (100 μl, Agilent Technologies) and separation was carried out at a flow rate of 400 nl.min−1 on a Peptide ES-C18 column (15 × 0.1 mm, 2.7 μm; Sigma-Aldrich) using a linear binary gradient (solvent A: 0.1% formic acid (FA); solvent B: 80% ACN 0.1% FA). MS and MS/MS analyses were performed online, in data-dependent mode with automatic switching between MS and MS/MS. Full scan MS spectra (300–1500 m/z) were acquired at 30,000 (m/z 400) resolution. Internal mass calibration was performed using Cyclomethicone (m/z 371.101230) as lock mass. Dynamic exclusion was enabled with exclusion size list of 500 and exclusion duration of 90 s. The eight most intense precursors were selected for subsequent fragmentation with normalized collision energy of 35%. Fragmentation spectra were acquired in the ion trap with an isolation window of 2.0 m/z, a target value of 1000, an activation Q of 0.25 and an activation time of 30 ms.CID spectra were processed in an in-house Mascot server (Version 2.1, Matrix Science, www.matrixscience.com, London, UK) using Proteome Discoverer (version 1.4.0.288, Thermo scientific) by searching against the NCBInr database using the taxonomy viridiplantae (http://www.ncbi.nlm.nih.gov) downloaded on June 06, 2014 and containing 40,910,947 sequences. The searches were performed with the following parameters: used enzyme: trypsin, 2 missed cleavages, mass accuracy precursor: 10 ppm, mass accuracy fragments: 0.8 Da, fixed modifications: Carbamidomethyl (C), dynamic modifications: Dioxidation (W), Gln->pyro-Glu (N-term Q), Glu->pyro-Glu (N-term E), Oxidation (HW), Trp-> Kynurenin (W). Identifications were filtered using the following settings; high peptide confidence (minimum confidence: 95%, peptide decoy database search: Target FDR (Strict): 0.01; Target FDR (Relaxed): 0.05, Validation based on: q-Value), with minimum two peptides per protein. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., ) via the PRIDE partner repository with the dataset identifier PXD001927 and 10.6019/PXD001927. Finally, all identifications obtained based on the NCBI database were matched on the Medicago truncatula reference genome using an online platform available at http://plantgrn.noble.org/LegumeIP/v2/blasttranscript.jsp. Sequences were imported as FASTA sequences and blasted to the Mt4.0v1_GenesCDSSeq_20130731_1800 database. Searches were performed with an E-value cut-off of 1e-04 and blast results were accepted in case more than 50% identity was reported. [...] Proteins were considered to be secreted in case the 2 servers SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP) and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP) predicted the presence of a signal peptide cleavage site and an extracellular location with the standard search parameters. […]

Pipeline specifications

Software tools Mascot Server, Proteome Discoverer, SignalP, TargetP
Databases ProteomeXchange LegumeIP
Applications MS-based untargeted proteomics, Protein sequence analysis
Diseases Multiple Sclerosis
Chemicals Amino Acids, Calcium Chloride, Egtazic Acid, Lithium Chloride