HPLC enrichment/isolation of proteins for post-translational modification studies from complex mixtures

The paper describes a macroporous RP-HPLC method for separation and isolation/enrichment of proteins from complex mixtures. The method is robust and efficient; using 2.1 or 4.6 mm diameter columns provides sufficient material for subsequent proteomic analysis. The main advantage of the method is that most protein variants are isolated in the same fraction, as separation is not based on differences in isoelectric point. This is highly advantageous for studying complex mixtures and post-translational modifications. Examples related to glycosylation analysis are discussed in detail

Changes of protein glycosylation in the course of radiotherapy

This is the first study of changes in protein glycosylation due to exposure of human subjects to ionizing radiation. Site specific glycosylation patterns of 7 major plasma proteins were analyzed; 171 glycoforms were identified; and the abundance of 99 of these was followed in the course of cancer radiotherapy in 10 individual patients. It was found that glycosylation of plasma proteins does change in response to partial body irradiation (~60 Gy), and the effects last during follow-up; the abundance of some glycoforms changed more than twofold. Both the degree of changes and their time-evolution showed large inter-individual variability

Fragmentation characteristics of glycopeptides

Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, “B” type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna- and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant