Biological material and growth conditions
Medicago truncatula cv Jemalong 5 seeds were surface sterilised and germinated at 27°C in the dark on 0.7% sterile agar . Two-day old seedlings were then transplanted into 400 mL plastic pots containing a mix of sterile soil of Epoisses and sand (1:2 v/v). Mycorrhizal inoculation was realized by adding Epoisses soil-based inoculum (spores, roots and hyphae) of the AM fungus Rhizophagus irregularis DAOM 181602 (formerly known as Glomus intraradices) . Seedlings (3 per pot) were grown for 4 weeks under controlled conditions (16 h photoperiod, 23°C/18°C day/night, 60% relative humidity, 220 μEinstein m-2.s-1 photon flux density). Control and R. irregularis-inoculated plants were watered each day with demineralised water and twice a week with a nitrogen-enriched nutrient solution. At harvest, roots were removed from their substrate, gently rinsed with deionised water, deep frozen, and stored at -80°C for later protein extraction.
Microsomal protein extraction
Microsome extraction of M. truncatula roots was performed at 4°C and obtained by differential centrifugation as previously described by Stanislas and co-authors . Briefly, roots were homogenized using a Waring Blendor in grinding buffer (50 mM Tris-MES, pH 8.0, 500 mM sucrose, 20 mM EDTA, 10 mM DTT and 1 mM PMSF). The homogenate was centrifuged at 16,000xg for 20 minutes (rotor JA 14 Beckman, CA, USA). After centrifugation, supernatants were collected, filtered through two successive meshes (63 and 38 μm), and centrifuged at 96,000xg for 1 h (rotor 45 Ti, Beckman). Pellets, representing the microsomal fraction, were resuspended in 10 mM Tris-MES, pH 7.3 250 mM sucrose, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μg/ml aprotinin and 10 μg/ml leupeptin. Protein amount was measured using the 2-D Quant Kit (GE Healthcare, Little Chalfont, UK).
In-solution protein digestion
Amicon Ultra-4 10 K centrifugal devices (Millipore, Bedford, MA, USA) were used for in-filter protein digestion. In the filtration devices, proteins (50 μg) from each control and mycorrhized plants were mixed with 10 mM DTT in solution A (8 M urea in 0.1 M Tris–HCl, pH 8.5) for 20 minutes to reduce protein disulphide bonds, and the excess reducing solution eliminated by centrifugation at 5,000xg for 40 minutes (Sanyo MSE Harrier 18/80, Japan). The protein sample was further cleaned by rinsing with 200 μl of solution A and repetition of the centrifugation step at 5,000xg for 40 minutes. One hundred microliters of 50 mM iodoacetamide in solution A were added on the filter and the filter was incubated in the dark at room temperature (RT) for 30 minutes followed by a centrifugation at 5,000xg for 30 minutes. Afterwards, 100 μl of solution B (8 M urea in 0.1 M Tris–HCl, pH 8.0) was added on the filter to adjust the pH, and centrifuged again. After repeating the latter step twice, trypsin (Trypsin Gold, mass spectrometry grade, Promega, Madison, WI, USA) was added in 30 μl of triethylammonium bicarbonate (TEAB) to an enzyme/protein ratio of 1:100. Protein digestion was carried out overnight at RT. Finally the peptides were collected by centrifugation of the filter units at 5,000xg for 40 minutes.
iTRAQ peptides labelling
Each tryptic digest was either labelled with iTRAQ reagent 114 or 117 following the manufacturer's instructions (AB SCIEX, Foster City, CA, USA). Control and mycorrhized samples were labelled by alternating 114 and 117 tags to avoid labelling bias. Some experiments were labelled with single use of one iTRAQ tag (114, 115, 116 and 117) targeting an overview of the iTRAQ 4-plex effect on peptide pI. iTRAQ reagents were dissolved in 70 μl of ethanol and added to the protein digest. After 1 h of incubation at RT, equal amounts of the different samples were pooled and concentrated by evaporation using a SpeedVac (Heto, Saskatoon, SK, Canada). The excess of iTRAQ reagents was removed by desalting the labelled peptides using C18 columns Supelco (DiscoveryTM DSC-18, 1 ml, 100 mg, Supelco Bellefonte, PA, USA). Peptides were eluted in 50% ACN (v/v), 0.1% TFA (v/v) and subsequently dried in SpeedVac (Heto) prior to peptides OGE fractionation.
3100 OFFGEL Fractionator and OFFGEL Kit pH 3–10 (Agilent Technologies, CA, USA) with 12 wells setup were used. Peptides were diluted in 1.8 ml of the focusing buffer containing only 5% (v/v) of glycerol in deviation from the supplier’s protocol. IPG strips were rehydrated by adding 40 μl of peptide IPG strip rehydration solution per well for 15 minutes. Then, 150 μl of sample were loaded in each well. Peptide focusing was performed until it reached 20 kVh with a maximum voltage of 8,000 V and maximum current of 50 μA. After focusing, the 12 peptide fractions were withdrawn and wells rinsed with 150 μl of H2O/MeOH/TFA (49/50/1 v/v) for 15 minutes. Rinsing solutions were pooled with their corresponding peptide fractions and concentrated in SpeedVac (Heto) prior to LC-MS/MS analysis.
The dried peptides were re-dissolved in 25 μl 0.1% TFA (v/v). Peptide separation was performed using an Ultimate 3000 nano LC system (Dionex, Sunnyvale, USA) equipped with a C18 column (PepMap 100, 3 μm, 100 Å, 75 μm id x 15 cm, Dionex) and connected to a Probot microfraction collector (Dionex). The mobile phase consisted of a gradient of solvents A 2% ACN (v/v), 0.2% TFA (v/v) in water and B 80% ACN (v/v), 0.08% TFA (v/v) in water. Peptides were separated at a flow rate of 0.3 μL/minute using a linear gradient of 60 minutes of solvent B from 0 to 5% in 5 minutes, followed by an increase to 30% in 5 minutes and to 65% in 30 minutes. The column was washed with 95% of solvent B for 5 minutes followed by regeneration with solvent A. Column effluent was mixed with MALDI matrix α-cyano-4-hydroxycinnamic acid (CHCA) and collected at a frequency of one spot every 30 seconds on an Opti-TOF LC/MALDI insert blank plate (AB SCIEX). MALDI plates were analyzed with a MALDI-TOF/TOF 4800 Proteomics Analyzer (AB SCIEX). The instrument was calibrated using the 4700 mass standard calibration kit (AB SCIEX). MS spectra between m/z 900 and 4,000 were acquired for every spot using 1,500 laser shots. The 8 most intense ion signals per spot having a S/N > 30 were selected as precursors for MS/MS acquisition.
Peptide and protein identifications were performed with the ProteinPilot™ Software 4.0.8085 revision 148085 (AB SCIEX) using the Paragon algorithm. Combined data and spectra from each OGE fraction were searched against the NCBI viridiplantae database (released on the 5th of May 2011) and a EST database of M. truncatula (http://www.medicago.org, released on the 18th of July 2011). The following search parameters were selected: iTRAQ 4-plex peptide label, cysteine alkylation, trypsin specificity, ID focus on biological modifications, and processing including quantitation and thorough ID. We only report protein identifications with a total ProtScore >1.3, which represents >95% statistical confidence in ProteinPilot. Proteins having at least one peptide above 95% of confidence were recorded.
MarvinSketch Calculator Plugin (http://www.chemaxon.com/marvin/sketch) , was implemented in this study to overcome the main bottleneck of the current available pI calculator such as the pI/MW tool of the ExPASy Proteomic Server (http://www.expasy.org) not giving the opportunity to calculate the pI of chemically modified peptides and consequently the pI of iTRAQ labelled peptides. This tool has been used to calculate pI of unlabelled and iTRAQ labelled peptides to explain some experimentally observed pI shifts.