Elsevier

Analytical Biochemistry

Volume 393, Issue 1, 1 October 2009, Pages 48-55
Analytical Biochemistry

Optimization of digestion parameters for protein quantification

https://doi.org/10.1016/j.ab.2009.05.050Get rights and content

Abstract

We present a rapid and efficient in-solution enzymatic digestion protocol suitable for mass spectrometry-based absolute protein quantification techniques. The digestion method employs RapiGest SF (an acid-labile surfactant), an excess amount of modified trypsin (enzyme-to-substrate ratio of 2.5:1), and an incubation time of 2 h. No reduction/alkylation reagents are used. Digestion parameters were varied systematically to monitor their effect on rate and completeness of digestion. To demonstrate the general applicability of the method, the optimization was done using a viral hemagglutinin (HA) as a model protein and then applied to ricin, a potent protein toxin extracted from the castor bean (Ricinus communis). The parameters that were optimized included incubation time, concentration of RapiGest SF, enzyme-to-substrate ratio, and incubation temperature. The optimization was done by comparing the yields from two protein-specific peptides originating from two different sites of the HA protein. The analysis was performed by liquid chromatography–tandem mass spectrometry in multiple reaction monitoring mode using isotopically labeled peptide standards for 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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