High Density Immobilized Trypsin in a Spin Column Format for Rapid Proteolysis and Increased Sequence Coverage

Tryptic digestion of proteins is one of the critical steps in any proteomics analysis and may heavily impact the quality of the data generated from the sample. The peptides generated are analyzed by mass spectrometry and used to identify and characterize the proteins in the sampled population. Several factors have been found to influence the results of proteolysis and the subsequent analysis. Selecting an inappropriate denaturant for a protein mixture can result in poor digestion, and ultimately in low sequence coverage, lowering the confidence of protein identification. In this study, the effects of denaturant selection on digestion efficiency were examined using both a high-density immobilized Trypsin Spin Column and traditional solution digestion. The spin column format allowed complete protein digestion in 15 minutes without concomitant generation of tryptic autolytic fragments, resulting in greater sequence coverage for individual proteins even in a complex mixture. A model system comprised of several proteins isolated from human serum was prepared and denatured in a variety of buffered systems. After reduction and alkylation, the protein solutions were digested utilizing the Trypsin Spin Column. Control digestions were performed overnight in solution with proteomics-grade trypsin. The digests were analyzed by SDS-PAGE and mass spectrometry to determine effectiveness of digestion and sequence coverage of the various proteins. The Trypsin Spin Columns, coupled with optimal denaturant, provided extremely rapid and complete digestions, resulting in greater sequence coverage in a much shorter time than traditional solution digestion.