Optimization of digestion parameters for protein quantification
Section snippets
Materials
Lyophilized bar-headed goose reagent was provided by the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration (Silver Spring, MD, USA). The reagent is a formalin-inactivated whole influenza virus prepared from strain A/bar-headed goose/Qinghai Lake/1A/05. The reagent, a lyophilized solid, was stored at –20 °C. The reagent working solution was prepared by adding 1.0 ml of 0.02% RapiGest SF in pure high-performance liquid chromatography (HPLC)-grade water. The
Results and discussion
The focus of this work was to develop a rapid and efficient digestion method suitable for MS-based protein quantification techniques. Digestion parameters, including time of incubation with trypsin, amount of trypsin, temperature of incubation with trypsin, and amount of an acid-labile detergent, were closely monitored for the purpose of understanding how they affect rate and completeness of digestion. The optimization of the digestion parameters was done using viral HA [24] from an H5N1 strain
Conclusions
Verification that complete digestion has been achieved is essential for accurately quantifying proteins using MS. However, the means to verify complete digestion are not trivial due to limited availability of reference protein materials. The work described in this article illustrates how digestion parameters can be optimized to maximize digestion efficiency of a protein. This method was demonstrated on two unrelated proteins, and it is more efficient in both time and completeness of digestion
Acknowledgments
We acknowledge the Center for Biologics Evaluation and Research of the U.S. Food and Drug Administration for providing valuable hemagglutinin samples used in this study. Reference in this article to any specific commercial products process service, manufacturer, or company does not constitute an endorsement or a recommendation by the U.S. government or the Centers for Disease Control and Prevention. The findings and conclusions reported in this article are those of the authors and do not
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