Developing Peptide Maps
For the Structural Characterization of Proteins
Peptide mapping is one of the most fundamental tools for analyzing the structure of proteins. It has been continuously enhanced over decades of use to increase sensitivity, discriminatory power, and information content. The current state-of-the-art technology is mass spectrometry, usually coupled to reversed phase HPLC. In this seminar, we will describe the steps and options for optimizing this process. Considerations in making the digest and otherwise preparing the sample will be described. The relative merits of different MS techniques will be considered, particularly in the context of analyzing fractionated versus unfractionated peptide mixtures. Where separation is required, the experimental parameters that can be manipulated to improve resolution will be described. Finally, we will show some new systematic approaches for optimizing these parameters and for selecting the best column size and gradient slope for a given sample.
Americas Operations
Waters Corporation, Milford, Massachusetts
Thomas E. Wheat earned his Bachelors of Arts in Biology from the College of St. Thomas and his Ph.D. in Cell Biology from the University of Illinois at Urbana-Champaign. He held research positions at Northwestern University before joining Waters Corporation . At Northwestern, his research involved protein purification and structure determination as applied in reproductive immunology. Previous positions at Waters include Senior Applications Biochemist, Life Sciences Laboratory Manager, and Field Marketing Manager for Mass Spectrometry and Autopurification Systems. He has recently been involved with the development of several novel techniques for purification and isolation, notably At-column Dilution. He received the Waters President's Club Award for Technical Excellence in 2000 and 2001. He has published and/or presented over 100 papers in the fields of analytical biochemistry, capillary electrophoresis, mass spectrometry and preparative chromatography. Numerous presentations have been made worldwide at scientific meetings and other public forums.