Lyophilized tear proteins (25-μg total protein) were reconstituted, denatured, and reduced in 50 mM ammonium bicarbonate solution (filter-aided sample preparation [FASP] kit; Expedeon, Inc., Harston, UK) 2% SDS solution (Vivantis Technologies, Selangor, Malaysia), and
tris(2-carboxyethyl)phosphine (TCEP) from iTRAQ kit (AB Sciex, Framingham, MA, USA) for 1 hour at 60°C. The reduced protein samples were then transferred to 30 kDa cut-off membrane cartridge (FASP kit; Expedeon, Inc.) to remove the excess reagent using 75% urea solution. The reduced samples were then alkylated using methyl methane thiosulfonate (MMTS; AB Sciex) for 20 minutes at room temperature. The alkylated-samples were further washed with 75% urea solution and 50 mM ammonium bicarbonte solution prior to trypsin digestion overnight with substrate: enzyme ratio of 1:25 at 37°C. Peptides were then eluted with 50 mM ammonium bicarbonate solution and sodium chloride (provided in FASP kit; Expedeon, Inc.). The eluted samples were lyophilized and tear samples were labeled with iTRAQ reagents for 3 hours at room temperature as follows: preoperatively collected tears with iTRAQ 114, 1 week postoperatively collected tears with iTRAQ 115 and 3 month postoperatively collected tears with iTRAQ 117 (
Fig. 1).
Labeled samples (iTRAQ 114, iTRAQ 115, iTRAQ 117) were then pooled together, dried and desalted using ultramicro spin columns (The Nest Group, Inc., Southboro, MA, USA) prior to nano-LC/MSMS analysis.
The one dimensional nano LC-MS/MS system (Dionex Ultimate 3000 Nano LC system; Thermo Fisher Scientific, Sunnyvale, CA, USA), coupled with AB Sciex TripleTOF 5600 system (AB Sciex), was used for the proteomic analysis. A 50 cm × 75 μm (internal diameter) Dionex Acclaim PepMap RSLC C18 packed column was employed (Thermo Fisher Scientific). This column was connected to a spray tip (New Objective, Inc., Woburn, MA, USA), which was directly coupled with the nanospray interface of AB Sciex TripleTOF 5600 MS. Samples were loaded onto a trap column (Dionex Acclaim PepMap 100 C18, 2 cm × 75 μm i.d.; Thermo Fisher Scientific) at a flow rate of 5 μL/min. After a 3 minute wash with loading buffer (2/98 vol/vol of acetonitrile [ACN]/water with 0.1% formic acid) the system was switched into line with the C18 analytical capillary column. A step linear gradient of mobile phase B (2/98 vol/vol of water/ACN with 0.1% formic acid) starting from 7% to 24% for 57 minutes, to 24% to 40% for 27 minutes, to 40% to 60% for 7 minutes, and 60% to 95% for 1 minute at a flow rate of 300 nL/min was used for this analysis. The typical parameters for TripleTOF 5600-MS were as follows: ionspray voltage floating (ISVF) = 2400 V, curtain gas (CUR) = 30, ion source gas 1 (GS1) = 12, interface heater temperature (IHT) = 125°C, declustering potential (DP) = 100 V. All data was acquired using information-dependent acquisition (IDA) mode with Analyst TF 1.5 software (AB Sciex). Time-of-flight mass spectrometry (TOF-MS) scan (experiment 1) parameters were set as follows: 0.25 seconds TOF-MS accumulation time in the mass range of 350 to approximately 1250 Da followed by product ion scan (experiment 2) of 0.05 seconds accumulation time in the mass range of 100 to approximately 1500 Da. Switching criteria were set to ions greater than m/z = 350 and smaller than m/z = 1250 with charge state of 2 to 5, and an abundance threshold of greater than 120 counts per second. Former target ions were excluded for 12 seconds and former ions were excluded after one repeat. Maximum number of candidate ions per cycle was 30 spectra. Information-dependent acquisition advanced “rolling collision energy (CE)” and “adjust CE when using iTRAQ reagent” were required.
The data was processed and searched against the IPI Human version 3.77 protein database (115194 proteins searched) using ProteinPilot software 4.1 (AB Sciex). Some important settings in ProteinPilot were configured as follows: (1) sample type: iTRAQ 4plex (peptide labeled), (2) Cys alkylation: MMTS; (3) digestion: trypsin, (4) instrument: TripleTOF 5600, (5) special factors: none, (6) identification focus: biological modifications, (7) search effort: thorough identification, and (8) bias correction and background correction were applied. Ninety-five percent confidence level was used at the peptide level. False discovery rate (FDR) analysis in the ProteinPilot software was performed and FDR less than 1% was set for protein identification. Reverse database search strategy was used to calculate FDR for peptide identification. For relative quantification, ProteinPilot software uses Pro Group algorithm to calculate the reporter ions' (iTRAQ114, 115, and 116) peak areas. Auto bias correction was applied to eliminate possible pipetting error during sample preparation.