Regardless of the type of analysis performed (identification, identification + quantification, targeted analysis), it is possible to search for post-translational modifications (PTMs) on proteins.
PTMs are covalent modifications of proteins, often resulting from enzymatic reactions, which can range from the addition of small chemical groups to complex macromolecules. PTMs contribute to the regulation of protein function, thus significantly increasing the functional diversity of the proteome.
PTMs can be detected by mass spectrometry because they involve a mass adduct on the peptides carrying them. For example, a peptide carrying phosphorylation will have a mass of +80 Da compared to its non-phosphorylated version.
However, it is important to note the following points when considering PTM analysis:
- In a global proteomic analysis (non-targeted), it is preferable to limit the search to a maximum of 3 to 5 different PTMs because increasing the number of possible combinations can generate a greater number of false positives in the analysis.
- It is often necessary to enrich modified peptides using specific antibodies (e.g., PTMscan kits – NEB; HighSelect – Thermo) because the stoichiometry of modified peptides is often low compared to unmodified peptides. In this case, analysis is generally performed with and without enrichment.
- Some modifications may be more complex to analyze, such as glycosylations, which have significant weight and different branches, making their molecular weight uncertain. Specific methods can be applied.
- In some cases, it is possible to validate that a peptide is modified without being able to specify exactly which amino acid carries the PTM. However, it is possible to perform different types of fragmentation in the mass spectrometer to retain PTMs on fragments and thus increase the possibilities of exact localization.
Feel free to consult us to assess the feasibility of your project and define the appropriate strategy.