Recent Advances in the Application of UPLC to LC/MS Peptide Mapping

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Thomas E. Wheat, Kim Van Tran, Beth L. Gillece-Castro, and Diane M. Diehl
Analytical Chemistry Group 9th Dansak Symposium, August 19 - 20, 2008, Denmark
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The analysis of protein structure is routinely based on enzymatic digestion to more manageable fragments. The separation of these fragments, peptide mapping, has long been a fundamental tool for structural analysis. Reversed phase HPLC has become the preferred tool for peptide mapping. The chromatographic separation is coupled to mass spectral detection for identification of the chromatographic peaks. The analysis of authentic protein samples is, however, more complicated because all samples are mixtures of native and modified or damaged proteins. Low abundance peptides, representing these trace modifications, may co-elute with major components. The analysis benefits therefore from the use of the most highly resolving chromatographic techniques. UPLC® peptide mapping routinely gives higher resolution than is possible with HPLC. UPLC is used in different ways to meet the requirements of specific analyses. First, the inherently high resolution can be used to ensure that all components in a sample are detected. Second, the high resolution can be used to reduce run time while preserving the resolution of a well-established map. Third, a very fast assay for particular diagnostic peptides can be developed. All three strategies will be illustrated with protein digests. The various options for improving selectivity, including gradient slope, temperature, and mobile phase modifier will be tested. Several new UPLC packing materials will also be compared in terms of their effects on selectivity in peptide maps. The systematically optimized chromatography improves the overall sensitivity and dynamic range of MS characterization, thus allowing accurate quantitation of modified proteins.

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