Posttranslational Modifications - Identification and localization of PTMs
Post-translational modifications (PTMs) are known to play a crucial role in almost all biological processes and they are involved in many diseases, such as cancer. However, only a few of the over 300 currently known modifications are extensively investigated so far. The most commonly studied PTMs in LC-MS-based proteomics include:
· Disulfide bridge formation
The reason why these PTMs are better studied than others is NOT because they are biologically more relevant, but rather because successful PTM-enrichment protocols have been established or because the stability and fragmentation characteristics of certain PTMs match more closely the proteomic sample preparation and MS instrumentation capabilities than others. Typical enrichment protocols for phosphorylated peptides include the usage of TiO2 or IMAC beads. As a result of the added negative charge of the phosphate group, modified phospho-peptides can be considerably enriched compared to non-modified peptides by ion‑exchange (IEX) chromatography. Separations on the basis of hydrophilic interactions (HILIC) are also able to enrich phosphorylated and certain other modified peptides. Further, PTM-specific antibodies have been developed, for example acetylated peptides can be enriched using an acetyl-Lys-specific antibody or phosphorylated tyrosine can be enriched with corresponding phospho-Tyr-specific antibody.
PTMs can be identified by mass spectrometry using the same gel-based or gel-free LC-MS shotgun approaches that are used for non-modified peptides, followed by a database search analysis specified for the PTMs of interest. Since post-translationally modified proteins and peptides are often present at low stoichiometry level, it is generally necessary to enrich for the PTM of interest in order to make these proteins and peptides detectable. To facilitate PTM localization, i.e. to pinpoint the exact amino acid where a specific PTM is attached, often a combination of different MS fragmentation techniques (CID, HCD, ETD, EThcD) has to be applied. Also the use of various different proteases for protein digestion is common in PTM projects, because the protein sequence coverage can be increased by the use of proteases with different cleavage-site properties.
The final outcome of a shotgun-based proteomic PTM analysis is typically a list modified peptide sequences, in which the nature of the modification usually needs to be defined upfront and the sample often has been enriched for this specific PTM or for the protein(s) of interest. For every PTM-carrying peptide not only a probability score gets computed, which serves as a measure for the likelihood that the given peptide sequence is correctly identified, but also a localization score for the several optionally modified amino acid positions gets provided.