Differential Gel-Based Proteomic Approach for Cancer Biomarker Discovery Using Human Plasma - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 225

limitations of 2D PAGE by increasing the quantitative accuracy of
detecting spot-to-spot differences ( 10, 18 ). To accelerate the dis-
covery of fundamental biomarker candidates in clinical samples,
this chapter describes a processing method for plasma samples that
facilitates the comparison of healthy donor and HCC patient
plasma proteomes using 2D DIGE, narrow pH strips, and nanoLC
tandem mass spectrometry (nanoLC-MS/MS) ( 17 ).

2. Materials

1. Blood collection tube: K 2 -EDTA 7.2 mg BD Vacutainer ® (BD

Bioscience, San Diego, CA, USA).

2.1. Preparation and
Pretreatment of
Clinical Samples

2. HPLC system, e.g., HP1100 LC system (Agilent Technologies,

Palo Alto, CA).

3. Multiple Affi nity Removal System (MARS): LC column

(Agilent Technologies; 5185-5984), Buffer A (Agilent
Technologies; 5185-5987), Buffer B (Agilent Technologies;
5185-5988).

4. Protease inhibitor (Complete Protease Inhibitor Cocktail,

Roche, 11 697 498 001, 20 tablets): Dissolve one tablet con-
taining protease inhibitors (antipain, bestatin, chymostatin,
leupeptin, pepstatin, aprotinin, phosphoramidon, and EDTA)
in 2 mL of distilled water.

5. Amicon Ultra-15 (5-kDa molecular weight cutoff; Millipore,

Barcelona, Spain).

6. 50% (w/v) or 6N trichloroacetic acid (TCA).

7. Lysis buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM

Tris-HCl (pH 8.5) (see Note 1).

8. pH indicator strip.

9. Protein assay: 2-D Quant Kit (GE Healthcare, Piscataway, NJ,

USA) or similar assay.

1. CyDye reagent: CyDye DIGE Fluor minimal dye (GE

Healthcare). Dissolve each dye to 400 pmol/

2.2. Components for
2D DIGE and 2D PAGE μ L in dimethyl-

formamide. Store as 1- μ L aliquots in individual tubes at −85°C

until use.

2. IPG strip: Immobiline Dry Strip, pH 3.5-4.5, pH 4.0-5.0, pH

4.5-5.5, pH 5.0-6.0, pH 5.5-6.7, pH 7-11, pH 3-10 NL,
pH 4-7, 24 cm long, 0.5 mm thick (GE Healthcare).

3. Quenching solution: 10 mM lysine.

4. Sample buffer: 7 M urea, 2 M thiourea, 4% (w/v) 3-((3-chol-

amidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS),

65 mM DTT, 30 mM Tris-HCl (pH 8.5), trace bromophenol
blue (BPB).