Trypsin trypsin gold mass spectrometry grade

Lyophilized MP4 protein, isolated by Bohn & Winckler (1991b) (link), was dissolved in 50 mM NH₄HCO₃ and the concentration was measured using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Then, 2 μL (1.5 μg) was diluted with 8 μL 5% methanol. Proteins were unfolded and reduced using 0.66 μL 0.5% RapiGest SF solution (lyophilized sodium-3-((2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl)-1-propane-sulfonate, Waters, Milford, MA, USA) and 0.52 μL 200 mM DTT solution (Fluka Chemie GmbH; Sigma-Aldrich^®, Zwijndrecht, Netherlands) for 30 min at 60 °C. Proteins were alkylated using 3.87 μL 200 mM NH₄HCO₃ solution and 1.05 μL 200 mM IAA solution (Fluka Chemie GmbH; Sigma-Aldrich^®, Zwijndrecht, Netherlands) for 30 min at room temperature in dark. Digestion was performed by adding 0.5 μL 50 ng/μL Trypsin/Lys-C Mix (Mass Spec Grade; Promega Corporation, Madison, WI, USA) for 60 min at 37 °C, followed by adding 0.5 μL 200 ng/μL Trypsin (Trypsin Gold, Mass Spectrometry Grade; Promega Corporation, Madison, WI, USA) for 120 min at 37 °C. Digestion was stopped by adding 0.5 μL formic acid, followed by 30 min incubation at 37 °C. Samples were centrifuged at 17,000×g for 30 min. All other reagents were purchased from Sigma-Aldrich^® (St. Louis, MO, USA).

Thirty-nine spots were excised automatically by Ettan Spot Picker (GE Healthcare). Gel plugs were picked from three different 2-DE gels where the selection of 2-DE images is based on the %vol of the spots on 2-DE images. For all spots, only one gel plug from one sample is excised. The protein gels were then reduced and alkylated using 100 mM DTT and 50 mM IAA, respectively. Gels were then incubated in Trypsin (+) solution [20 ng/µL trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega), 40 mM ammonium bicarbonate, 0.2 mM HCl, 5 mM calcium chloride (CaCl₂) and 10% acetonitrile (ACN)] for 5 min at R.T. Later, gels were incubated in Trypsin (−) solution containing the same components as Trypsin (+) solution but without trypsin at 37 °C overnight.
Following collection of supernatant, gels were incubated in ultra-pure water (Wako Pure Chemical Industries) at R.T. for 10 min. The supernatant was collected again and peptide extraction was carried out using 60% ACN, 80% ACN and 100% ACN. The collected peptide solution was concentrated by a centrifugal evaporator (CVE-2000, EYELA Tokyo Scientific Instruments) to approximately 10 µL.
The concentrated peptides were desalted using C-tip (AMR) before mass spectrometry analysis. This was performed according to the manufacturer’s guideline.

Proteins in rehydration buffer (described earlier) were exchanged to 0.5 M TEAB buffer (compatible for iTRAQ labeling) using Amicon Ultra 0.5 mL centrifugal 3 kDa filters (Millipore, Watford, UK). Following buffer-exchange protein concentrations were determined using QuickStart Bradford reagent (BioRad, USA) and quality was checked on a 12% SDS gel. In-solution digestion of respective protein samples (100 μg each); control (0 h) and treatments (40 h, 88 h, and 120 h) were performed using trypsin (trypsin Gold, mass spectrometry grade, Promega, Madison, WI, USA) at a ratio 1: 30 (trypsin: protein), following the manufacturer’s instructions. Four-plex iTRAQ labeling kit (AB Sciex UK Limited, UK) having labels 114, 115, 116 and 117 was used to label trypsin-digested peptides of 0, 40, 88 and 120 h FC2 samples respectively, following the manufacturer’s instructions. All the labeled peptides were pooled and proceeded for OFFGEL fractionation using a 3100 OFFGEL Fractionator (Agilent Technologies, Santa Clara, CA) with high resolution (pH 3–10, 24 cm) IPG strips. Fractions were collected and enriched using Zip-Tip C18 pipette tips (Millipore, USA).