Optimizing Trypsin Digestion Parameters for Plasma Proteins

TrypsinLysC Page 2Biomarkers in biological fluids in particular have the potential to inform regarding risk of disease or to allow early detection for more effective treatment. Plasma/serum is considered the universal source of biomarkers. This fluid is, indeed, easily collected, and the important point is that plasma collects proteins from each and every tissue, compared to other fluids such as urine or cerebrospinal fluid. Optimizing experimental conditions (i.e., use of trypsin for the digestion of target proteins) used to discover or monitor biomarkers in plasma is critical to successful detection of biomarkers.

In a recent publication by Proc et al., plasma denaturation/digestion protocols were compared using quanititation methods. In this reference 14 combinations of heat, solvent (acetonitrile, methanol, trifluoroethanol), chaotropic agents (guanidine hydrochloride, urea) and surfactants (sodium dodecyl sulfate (SDS)and sodium deoxycholate (DOC) with effectiveness in improving tryptic digestion. Digestion efficiency was monitored by quantitating the peptides from 45 moderate- to high-abundance plasma proteins using tandem mass spectrometry in multiple reaction mode with a mixture of stable isotope labeled analogues of these peptides as internal standards. In the results, Proc et al. noted that use of either DOC and SDS produced an increase in the overall yield of tryptic peptides. Since SDS is not compatible with mass spectrometry and DOC can be easily remove by acid precipitation, the overall recommendation was the use of DOC with a nine hour digestion procedure.

Literature Cited:

Proc, J.L. et al. (2010) A Quantitative Study of the Effects of Chaotropic  Agents Surfactants and Solvents on the Digestion Efficiency of Human Plasma Proteins by Trypsin J. Proteome Res. 9, 5422–37.

The following two tabs change content below.

Gary Kobs

Strategic Marketing Manager at Promega Corporation
Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

Leave a Reply