Overview: Post-translational modifications (PTMs) are changes made to proteins after they have been synthesized. PTMs are critical in controlling many biological processes, and examining their diversity is essential for understanding the mechanisms of cell regulation. Mass spectrometry (MS) is a widely used technique to analyze PTMs.
Sample Preparation: The sample preparation includes protein extraction, denaturation, reduction, and alkylation of cysteine residues. Proteins are then cleaved into smaller peptides using proteolytic enzymes such as trypsin. The sample preparation can differ depending on the PTM of interest. For example, the analysis of phosphorylation or other low abundant PTMs includes enrichment.
High-performance liquid chromatography – mass spectrometry (HPLC-MS) analysis: The resulting peptide mixture is separated using liquid chromatography (LC) to reduce sample complexity and improve sensitivity. The peptides are then ionized and introduced into the mass spectrometer. Peptides are separated on MS1 mass spectra based on their mass-to-charge ratio (m/z). Each peptide is further fragmented, and the resulting fragments presenting on MS2 mass spectra carry the sequence and PTMs information of the peptide.
Data analysis: The MS data are analyzed using Biopharma Finder or PMI Byos software tools to identify and localize the PTMs on certain proteins. The percentage of certain PTMs, such as deamidation, oxidation, can also be determined.