Optimization of diagonal chromatography for recognizing post-translational modifications

Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents a formidable analytical challenge. This paper examines the potential of diagonal chromatography for recognizing post-translational modifications. Diagonal chromatography is the process of using the same chromatographic separation in two dimensions. Between the two dimensions, a chemical modification is applied to all fractions. Substances that have been modified are revealed by the change in their chromatographic properties between the two dimensions. When the modification is specific to a particular type of post-translational modification, peptides that carry the modification have the potential to be revealed. Changes in the retention time of modified peptides have to be large enough to be different from unmodified peptides. Tyrosine, serine, and threonine phosphorylation were identified with diagonal chromatography. Heptafluorobutyric acid was used as an ion-pairing agent to improve the selectivity between serine and threonine phosphorylated peptides and parent peptides after dephosphorylation. The diagonal chromatography method was also examined in the recognition of glycopeptides. However, changes of retention time after deglycosylation were considered to be too small to make this an unequivocal method for the study of glycosylation.