Application of UPLC™ to Peptide Mapping

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Third Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries Practical MS
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The combination of MS detection with LC separation as a peptide mapping technique is fundamental to the characterization of proteins. Tandem MS techniques are useful for extracting structural information from incompletely resolved peptides, but the best sensitivity and the most readily interpretable spectra are associated with chromatographically resolved single peptides. This chromatographic resolution for a typical complex digest requires highly resolving chromatographic techniques. We describe here the use of very small particle packings, Ultra Performance Liquid Chromatography™, to maximize chromatographic resolution and sensitivity. The utility of this approach is evaluated based on the usability of the mass spectra obtained. The use of these high resolution maps for improved quantitative analysis, especially of trace peptides will be considered. We will give special attention to the analysis of peptide glycoforms.

Characterization of glycoproteins includes analysis of the primary structure of the protein and analysis of the attached oligosaccharides. The common glycan mapping techniques are used to establish the pattern of sugar structures associated with the protein but cannot reveal site-specific structures because the sugars are first released from the protein. In peptide maps, the intact glycopeptides are separated so that the structures of the oligosaccharides can be associated with a particular site of attachment. The glycopeptides, however, often give incomplete separations and distorted peaks. With chromatographic techniques based on sub-2µm particles with the chemical characteristics associated with ACQUITY BEH Technology™ packing materials, these structures are better resolved for more complete characterization.

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