Top-to-Bottom Protein Characterization
Pressurized pepsin digestion as an automatable alternative to trypsin
Workflow showing the integration of bottom-up and top-down proteomics. Enlarge Image.
Scientists from Pacific Northwest National Laboratory (PNNL) and Korea University have demonstrated that using pepsin digestion and integrating two strategies for in-depth characterization and quantitation of proteins—bottom-up and top-down—can provide more effective protein characterization in less time. Bottom-up proteomics provides the peptide sequences using mass spectrometry (MS) analyses after the peptides have been digested from the proteins. Top-down proteomics conversely entails MS analyses of intact proteins. The researchers showed how analyzing protein mixtures using an integrated strategy—including using EMSL’s 12-Tesla Fourier-Transform Ion Cyclotron Resonance mass spectrometer—reduces the total amount of sample required to obtain both the top-down and bottom-up data, increases throughput, and improves protein sequence coverage. Such in-depth characterization and quantitation of proteins in biological systems can contribute to advances in human health and understanding the progression of disease and can be applied in bioremediation. This research was featured on the cover of a special issue of Molecular & Cellular Proteomics in February 2011.
Data obtained from top-down and bottom-up workflows are complementary, so previous attempts to integrate the strategies used extensive fractionation of the intact protein separation followed by bottom-up analysis of the collected fractions. Previous researchers had considered an online digestion approach, but no available online integrated method was robust enough for high-throughput analyses, in part due to the choice of proteolytic enzyme used for the bottom-up segment—trypsin. Instead, for this study the scientists chose pepsin, which unlike trypsin is active in acidic conditions and therefore compatible with the online approach. Using a Barocycler or modified high-pressure liquid chromatography system referred to as a fast online digestion system (FOLDS), they applied high-pressure to pepsin to digest proteins. The scientists tested FOLDS using pepsin by analyzing whole protein extract from Shewanella oneidensis, a soil bacteria of great interest in bioremediation, and compared the results against traditional trypsin digestions on the same platform. FOLDS was subsequently modified with a trapping reactor to demonstrate an ultra-rapid, integrated top-down bottom-up proteomic strategy using a standard mixture of proteins and a monkey pox virus proteome.
Scientific Impact: The combined use of pepsin and pressure was demonstrated to reduce digestion times to minutes from the typical overnight reaction time, allowing the ultra-rapid characterization of proteins and peptides. The work represents a new and improved way for high-throughput analysis of complex biological systems.
Societal Impact: Rapid and comprehensive characterization of proteins and protein modifications and the measurement of absolute protein are relevant to human health and the progression of various disease states. This research also provides insights that are relevant to bioremediation efforts.
Reference: Lopez-Ferrer D, K Petritis, EW Robinson, KK Hixson, Z Tian, JH Lee, SW Lee, N Tolic, KK Weitz, ME Belov, RD Smith, and L Pasa-Tolic. 2011. “Pressurized Pepsin Digestion in Proteomics: An Automatable Alternative to Trypsin for Integrated Top-down Bottom-up Proteomics.” Molecular & Cellular Proteomics 10(2): M110.001479. DOI: 10.1074/mcp.M110.001479.
Acknowledgement: This work was supported by the National Institutes of Health (NIH) National Center for Research Resources, NIH National Cancer Institute, and PNNL’s Laboratory Directed Research and Development Program.
Released: March 16, 2011
