Optimization of Novel GELFrEE Fractionation for Molecular Weight–Based In-solution Protein Separation from Buffalo Meat, Pork, and Chicken

This study evaluates and compares the novel GELFrEE protein fractionation technique with OFFGEL electrophoresis in their performance as in-solution fractionation approaches for total and myofibrillar/sarcoplasmic proteins isolated from different food animals and poultry meat. The current study demonstrates that both OFFGEL and GELFrEE techniques are suitable for separation of relatively low-abundant proteins with an average of 0.125 p I width or 1.0 kDa resolution and effectively reduce the sample complexity in 24–36 h and 2–3 h, respectively. The 5%, 8%, and 12% GEFREE 8100 cartridges are effective in fractionating total buffalo meat proteins within a mass range of 10–240 kDa, 10–140 kDa, and 5–45 kDa, respectively. Our work also provides the usefulness of GELFrEE separation with an average fraction width of 40 kDa (5%), 10 kDa (8%), and 4.0 kDa (12%) for further complementary protein identification using MALDI-TOF MS. We have demonstrated a maximum protein loading capacity of 2.5 mg and 8.0 mg for OFFGEL and GELFrEE with up to 50–100% sample recovery for the latter. The performed comparison of the fractionation techniques demonstrates the potential of GELFrEE fractionator for rapid, reproducible, high degree of resolution and high sample recovery tool in proteomic applications.