Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

BACKGROUND: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. RESULTS: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). CONCLUSIONS: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.

Study on the enzymatic hydrolysis technology of oyster / 中国海洋药物

Objective To research the optimum hydrolysis conditions of oyster protein so as to raise the protein recovery and degree of hydrolysis.Methods Considering protein recovery,degree of hydrolysis,bitterness and clarity,the best enzyme was selected;Orthogonal test was designed to determine the best enzyme hydrolysis conditions;Based on hydrolysis and protein recovery,the effect of heat treatment,ultrasound and microwave processing and handling on enzymatic hydrolysis of oyster were investigated.Results and Conclusion Bromelain protease was suitable for the proteolysis of oyster,and the optimal conditions were:pH 6,temperature 55℃,substrate concentration 1∶3,E/S=800U/g,4 hours.In the optimal condition,the recovery of protein and degree of hydrolysis were 67.55%and 29.86% respectively in the hydrolysate.Heating,ultrasound technology and microwave treatments before hydro-lysis were harmful to enzymatic hydrolysis of oyster.