An Open Tubular CEC Column of Excellent Separation Efficiency for Proteomic Analysis

Experimental

Materials. Glacial acetic acid, trifluoroacetic acid (TFA), polyethylene glycol (PEG), sodium hydrogen phosphate, sodium dihydrogen phosphate, azobisisobutyronitrile (AIBN), 4-aminostyrene, ethyleneglycol dimetnacryalte (EDMA), γ-methacryloxypropyl trimethoxysilane (MAPS), lysozyme, ribonuclease A, α-chymotrypsinogen A, myoglobin, cytochrome C, and trypsin were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade methanol, acetonitrile (ACN), 2-propanol, acetone, and water were obtained from Mallinckrodt Baker (Phillipsburg, NJ, USA). Silica capillaries (50 mm ID, 365 mm OD) were purchased from Grace (Deerfield, IL, USA).

Instrument. CEC experiments were performed on an Agilent (Waldbronn, Germany) HP3DCE system with a diode array detector and the Chemstation data processing software. The stock buffer solution (50 mM phosphate) was prepared in distilled water and kept in a refrigerator. Later, the pH of phosphate buffer was controlled to the desired pH followed by acetonitrile addition to get the final mobile phase. All the samples and eluents were filtered through a 0.2 μm cellulose membrane before analysis. Samples were injected hydrodynamically for 4 s under a pressure of 5 mbar. The detection wavelength was set to 214 nm. All the separations were carried out at a constant CE voltage of −20 kV and a temperature of 25 ℃ throughout. The OT-MIP column was flushed by running the eluent for about 1 h to acquire the stable baseline.

Tryptic Digest and Protein Samples. Cytochrome C 2.5 mg was mixed with trypsin 1.0 mg, 2.0 M urea 500 uL, 0.1 M ammonium bicarbonate 500 uL, and incubated for 24 h at 37 ℃. Then it was quenched with 1 mL 0.1% TFA and stored in a freezer until analysis. The protein sample was prepared by dissolving 1.0 mg each of lysozyme, ribonuclease A, α-chymotrypsinogen A, myoglobin, cytochrome C in 1.0 mL water, and stored in a freezer. Later, it was diluted 1,000 times in the mobile phase and injected.

Preparation of OT-CEC Column. The fused silica capillary was modified according to the procedure published elsewhere.35-38 Briefly, the silica capillary of 50 cm length was treated with 1 M NaOH, washed with water, 0.1 M HCl, water, and acetone in sequence, and dried under a flow of N2. A solution composed of 4 μL MAPS in 1.0 mL of 6.0 mM acetic acid was filled in the capillary for 6 h and the capillary was flushed thoroughly with methanol and dried under a nitrogen flow. After some trial-and-error based optimization, a mixture was prepared for formation of OT stationary phase for separation of proteins as follows: PEG (MW 10,000) 4.9 mg, 4-aminostyrene 8.2 μL, EDMA 59 μL, AIBN 3.5 mg dissolved in 1.0 mL 9/1 (v/v) ACN/2-propanol. The mixture was sonicated for 10 min and purged with nitrogen for 10 min. A piece of silica capillary was filled with the mixture and both capillary ends were sealed with rubber plugs. The reaction was carried out for 4 h at 50 ℃, then the capillary was thoroughly flushed with ACN, 9/1 (v/v) methanol/acetic acid, 5/5 (v/v) methanol/water in sequence. The same procedure was repeated for preparation of OT-CEC columns for the tryptic digest (separation of peptides) with somewhat different formulation of reaction mixture. The optimum formulation was found as follows: PEG (MW 10,000) 9.8 mg, 4-aminostyrene 24.6 μL, EDMA 59 μL, AIBN 3.5 mg dissolved in 1.0 mL 9/1 (v/v) ACN/2-propanol.

Optimization of Separation of Tryptic Digest. In addition to the optimization of formulation of reaction mixture, the elution conditions were also optimized by varying pH and mobile phase composition.