The technique of peptide mapping provides significant and key information on PTMs. For peptide mapping studies, including investigations into the nature and location of PTMs, mass spectrometers such as the Waters Q-TOF type instruments are invaluable as they are able to generate fragment ion data from peptides as they enter the source in real time. These fragment ions give peptide sequence information and thus confirmation of the detected peptide, providing identification for peptides that are mass shifted as the result of PTMs. Furthermore, if the PTM is present at a sufficient level, the fragment ion information produced may be able to give the precise location of the PTM within the peptide sequence.
Whilst mass spectrometry can provide significant information on the nature and location of PTMs, there are techniques that can provide orthogonal data to support these assignments. The use of orthogonal procedures for determination of structural characteristics is highly regarded by regulatory authorities in any structural investigation. Examples are the use of imaged capillary isoelectric focusing (icIEF), which provides charge-based information and can help with, for example, investigations into the presence of C-terminal Lysine on monoclonal antibody heavy chains and, more generally, deamidation of proteins. Capillary gel electrophoresis (CE-SDS) can also serve to support mass spectrometry-based investigations into larger mass PTMs such as PEGylation, glycosylation and proteolytic processing.