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Biological Sciences Division
Research Highlights

April 2011

Top-to-Bottom Protein Characterization

Pressurized pepsin digestion as an automatable alternative to trypsin

MCP cover
High-pressure digestion combined with intact protein high-pressure liquid chromatography enables the integration of top-down and bottom-up proteomics for more complete characterization of protein isoforms arising from post-translational modifications. This research was originally published in Molecular & Cellular Proteomics. 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 Paša-Tolic. "Pressurized Pepsin Digestion in Proteomics: An Automatable Alternative to Trypsin for Integrated Top-down Bottom-up Proteomics." Molecular & Cellular Proteomics. 2011. 10(2): M110.001479. J© the American Society for Biochemistry and Molecular Biology. Enlarge Image

Results: You can have it both ways when it comes to proteomics. Scientists from Pacific Northwest National Laboratory and Korea University showed that using pepsin digestion and integrating bottom-up and top-down strategies for analyzing proteins can provide more effective protein characterization in less time with smaller sample amounts. Bottom-up proteomics provides the peptide sequences using mass spectrometry (MS) analyses after the peptides have been digested from the proteins. Conversely, top-down proteomics entails MS analyses of intact proteins.

Why it matters: 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.

Methods: 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. Previously, researchers had considered an online digestion approach, but no available method was robust enough for high-throughput analyses, in part because of the choice of proteolytic enzyme used for the bottom-up segment-trypsin.

Instead, for this study the scientists chose the digestive enzyme pepsin, which, unlike trypsin, is active in acidic conditions and therefore compatible with the online approach. Using a modified high-pressure liquid chromatography system referred to as a fast online digestion system (FOLDS), they applied high pressure to rapidly digest proteins using pepsin. The scientists tested FOLDS using pepsin by analyzing whole protein extract from Shewanella oneidensis, a soil bacterium of great interest in bioremediation, and compared the results against traditional trypsin digestions on the same platform. They subsequently modified FOLDS with a trapping reactor to demonstrate an ultra-rapid, integrated, top-down bottom-up proteomic strategy.

Acknowledgments: 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. The research team included Ljiljana Paša-Tolic, Robby Robinson, Kim Hixson, Zhixin Tian, Nikola Tolic, Karl Weitz, Mikhail Belov, and Dick Smith, PNNL; former PNNL staff members Daniel Lopez-Ferrer and Konstantinos Petritis; and Jung Hwa Lee and Sang-Won Lee, Korea University.  Portions of the work were performed at EMSL, a DOE national scientific user facility located at PNNL.

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 Paša-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.

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Bottom-up and top-down proteomics

Bottom-up proteomics is a shotgun approach where researchers use mass spectrometry to detect peptides—pieces of a protein—hundreds to thousands at a time. This leads to identifying the presence of proteins that comprise a cell, a tissue, or an organism.

Bottom-up analysis is an excellent technique for identifying a protein's presence—especially for simple systems like microbes-but not necessarily for identifying the form of the protein present. It also requires repeating the experiments enough times to obtain the statistically significant observation. At EMSL, it can take 4 hours per experiment.

A more recent approach is top-down analysis—an innovative method for helping researchers understand the role, function, structure, and changing nature of cellular proteins, particularly within more challenging organisms.

The approach enables analysis of intact proteins. It requires a high performance mass spectrometer, such as EMSL's 12-tesla Fourier-transform ion cyclotron resonance mass spectrometer. Combining bottom-up and top-down methods result in not only a scientific understanding of the protein, but expand applications to environmental challenges.