Coupled Application of Aqueous Two-Phase Partitioning and 2D-Electrophoresis for Characterization of Soybean Proteins

Abstract

A novel combination of 2-D electrophoresis with hydrophobic partitioning in aqueous two-phase systems (ATPS) was extended to an alternative ATPS and both systems used for the three-dimensional characterization of the proteins extracted from soybeans. The 3-D plots of molecular weight, isoelectric point, and surface hydrophobicity were obtained using two different phase-forming salts: Na₂SO₄ and potassium phosphate. Six proteins with known hydrophobicities were used to validate the ATPS-based method. Molecular properties obtained using the PEG-sulfate system resulted in a wider range of proteins characterized. The wide range of concentration and strongly hydrophilic character of the soy extracts limited the coverage obtained; reduction of the storage protein content aided detection. The number of proteins simultaneously and accurately characterized by this method as currently implemented is limited by the dynamic range of staining, the ability to quantify strongly partitioning proteins in both phases, and loss of proteins of limited solubility in the ATPS or during the removal of phase-forming components.

Keywords:

• 2-D electrophoresis
• aqueous two-phase systems
• protein characterization
• soybean

ACKNOWLEDGEMENTS

Authors wish to acknowledge the financial support of Tecnológico de Monterrey Biotechnology Research Grant CAT161. Funding for the work at Iowa State University was provided by the USDA CREES Grants #2006-34496-17122 and #2008-34496-19348. Also we thank Ryan Swanson from Iowa State University, for his independent confirmation of a test subset of the 2D gel matches.

Notes

^a Standard errors reported are the result of three independent measurements for TCA precipitation and duplicates for ATP partitioning.

^b Expressed as the percentage of the initial amount of protein added to the system (1.0 mg/g ATPS). The amount of protein at the interface was estimated as the necessary to complete 100% recovery.

^c Percentage relative to the amount of protein measured on top/bottom phase previous to precipitation step.

^d ATPS is PEG 3350 (14.8%)-potassium phosphates (10.3%)-NaCl (3%) at pH 7.

^e ATPS is PEG 3350 (15.7%)-Na₂SO₄ (8.9%)-NaCl (3%) at pH 7.

^f Mixture of LYS, BSA and LAC as model proteins.

^a Systems formulated at pH 7 and 25°C. Sample load was 1.0 mg protein/g ATPS.

^a All data are the average of duplicate experiments run at pH 7 and 25°C. Load of protein was 1.0 mg/g ATPS.

^b Amount of each protein present per 5 g ATPS, obtained as the sum of the amount of each protein quantified in top and bottom phases and considering losses during ATP partitioning and TCA precipitation steps for each sample.

^c Calculated using the sum of top + bottom matched protein divided by the total amount of the same protein spot matched in the gel where no partition experiment was performed (total extract) and considering TCA loss for each case.

^d Capital letters show presumed storage subunits commonly identified in both systems (see text).

^a All data are the average of duplicate experiment in the case of 2DE and triplicate for protein assay.

^b Protein load was 1 mg/g ATPS for soybean samples and model proteins mixture.

^c ATPS: PEG 3350 (14.8%)-potassium phosphate (10.3%)-NaCl (3%) and PEG 3350 (15.7%)-Na₂SO₄ (8.9%)-NaCl (3%) at pH 7.

^d Mixture: lysozyme, bovine serum albumin and α-lactoalbumin.

^e Calculated as the sum of proteins detected by spot densitometry in top divided by the sum of the proteins detected in bottom phase for each sample.

Total protein concentration (ppm): Top phase, 129.2; Bottom phase, 1215; Total soybean extract, 6671.

Total spots count: 83 *11 spots not found in either phase.

Partition coefficient: 0.11.

All data are the average of two experiments using two-phase system: 15.7% PEG 3350, 8.9% Na₂SO₄, 3% NaCl, pH 7, V_r = 1.25 at 25°C. Load of protein was 1.0 mg/g ATPS from a total soybean extract of 5.3 mg protein/mL. Protein concentration is expressed in parts per million in the ATPS. % Recovery calculated using the sum of top + bottom protein divided by the total amount of the same protein matched in the gel where no partition experiment was performed (total extract). Log K_p values for proteins not detected in top phase were estimated as the highest K_p possible taking the concentration of the faintest spot quantified as the limit of detection.

Total protein concentration (ppm): Top phase, 95.52; Bottom phase, 2218; Total soybean extract, 6671.

Total spots count: 97 *29 spots not found in either phase.

Partition coefficient: 0.04.

All data are the average of two experiments using two-phase system: 14.8% PEG 3350, 10.3% potassium phosphate, 3% NaCl, pH 7, V_r = 1.25 at 25°C. Load of protein was 1.0 mg/g ATPS from a total soybean extract of 5.3 mg protein/mL. Protein concentrations are expressed in parts per million in the ATPS. % Recovery calculated using the sum of top +bottom protein divided by the total amount of the same protein matched in the gel where no partition experiment was performed (total extract). Log K_p values for proteins not detected in top phase were estimated as the highest K_p possible taking the concentration of the faintest spot quantified as the limit of detection.