Protein Separation Using 2D-HPLC and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

The extracted and desalted mass proteins were separated on a 2D-HPLC system using an ion-exchange column for the first dimension and a reverse-phase monolithic column for the second dimension (Morisaka et al., 2012). The 2D-HPLC system consisted of PU-712 and PU-714 pumps (GL Sciences), two trap columns (Nacalai Tesque), a 7725 injector (Rheodyne), a MU-701 UV detector (GL Sciences), and a 10-port valve MV-790 (GL Sciences). Raw chromatographic ASCII formatted data files were collected with EZChrom Elite software (GL Sciences). The secreted proteins were separated into six (F1–F6) fractions for the first-dimension extraction in ion-exchange mode. An anion-exchange column, NUCLEOSIL 4000-7 PEI (MACHEREY-NAGEL), was used with a flow rate of 0.8 ml/min for the ion-exchange mode for the first-dimension extraction. A gradient was provided by changing the mixing ratio of two eluents: A, 20 mM Tris/HCl (pH 8.2) and B, 20 mM Tris/HCl (pH 8.2) containing 1 M NaCl. The gradient involved six steps (F1–F6) with 0, 10, 20, 30, 50, or 100% B for 3 min. Each fraction was loaded into the second-dimension column through a 10-port valve and separated in reverse-phase mode. In reverse-phase mode, a wide-pore monolithic column [5 cm length, 2.3 mm inner diameter (ID)] was used with a flow rate of 1.0 or 1.5 ml/min. A gradient was provided by changing the mixing ratio of two eluents: C, 0.1% (v/v) trifluoroacetic acid and D, acetonitrile containing 0.1% (v/v) trifluoroacetic acid. The gradient initially contained 10% B, which was increased to 60% B for 5 or 3 min, then increased to 95% B to wash the column, and finally restored to the initial conditions and maintained for re-equilibration of the column. Because no protein was detected in fractions from 0 to 2 min in the preliminary test, the fractions including separated proteins were collected only from 2 min to 4.5 min at 30-s intervals for each ion-exchange mode fraction (F1–F6). In total, 30 fractions were collected during 2D-HPLC analysis. These fractions were dried by vacuum centrifugation and re-suspended in 10 μl of phosphate-buffered saline (pH 7.4). The re-suspended samples were mixed with an equal volume of 2× sodium dodecyl sulfate (SDS) sample buffer, then heated at 100°C for 3 min prior to loading onto a gel for subsequent SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Tricine SDS-PAGE and Laemmli SDS-PAGE systems were used. The fractions from 2 to 3 min (total of 12 fractions) were separated using a Tricine SDS-PAGE system with 15% Tris-tricine gels (e-PAGEL; ATTO) and a standard minislab PAGE apparatus (model AE-6500; ATTO) to separate low-molecular-weight proteins. Low-Range Rainbow Molecular Weight Markers (GE Healthcare) were used to determine the molecular masses of the proteins. In addition, the fractions from 3 to 4.5 min (total of 18 fractions) were separated using a Laemmli SDS-PAGE system with 5–20% gradient gels (e-PAGEL; ATTO). Full-Range Rainbow Molecular Weight Markers (GE Healthcare) were used to determine the molecular masses of the proteins. The proteins were silver-stained using a Sil-Best staining kit (Nacalai Tesque), in accordance with the manufacturer’s instructions.