Systematic Development of Peptide Maps for Protein Characterization

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Thomas E. Wheat, Beth Gillece-Castro, Ziling Lu, Uwe D. Neue, and Jeffrey R. Mazzeo [Waters]
WCBP, January 24-27, 2006
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Peptide mapping using reversed phase HPLC has become the most generally useful tool used in the detailed characterization of protein structure. Methods development for this technique can be a difficult and time consuming process. Peptides representing the entire sequence of the protein must be separated as discrete peaks, and the map must accommodate all the important structural variations of the protein. We have evaluated the impact of the chromatographic column, the mobile phase chemistry, and the operating conditions on the separation of peptides. These experimental evaluations lead to a systematic approach to establishing robust and reliable protocols for peptide mapping. Detector selection will be considered because it constrains the mobile phase selection. Principles for choosing the column will be described, including bonded phase, pore size, and particle chemistry in addition to column dimensions, the initial experimental conditions, including an approach to selecting column dimensions, will be discussed. After examining the initial peptide map, the separation must be optimized. We will review the principles underlying the options for making the peptide map better. These factors include mobile phase modifier, modifier concentration, gradient slope, and temperature. The manipulations will be assessed based on altered selectivity. Using examples of complex digests of large and variously modified proteins, the chromatographic observations will be correlated with peptide structure where possible

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