Application of UPLC™ to Peptide Mapping
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