https://www.selleckchem.com/products/azd9291.html The enrichment strategies, together with quantification of two different PTM types from the same sample, allow investigation of the interplay of those two PTMs, which are important for signal transduction inside the cell (phosphorylation), as well as for messaging between cells through decoration of the cellular surface (glycosylation).The analysis of disease-related changes in the phosphorylation status of cellular signal transduction networks is of major interest to biomedical researchers. Mass spectrometry-based proteomics allows the analysis of phosphorylation in a global manner. However, several technical challenges need to be addressed when the phosphorylation of proteins is analyzed. Low-abundant phosphopeptides need to be enriched before analysis, thereby introducing additional steps in sample preparation. Consequently, the applied quantification strategies should be robust towards elaborate sampling handling, rendering label-based quantification strategies the methods of choice in many experiments. Here, we present a protocol for SILAC labeling and the subsequent isolation of phosphopeptides using TiO2 affinity chromatography. We outline the corresponding LC-MS/MS analysis and the essential steps of data processing.Quantitation using mass spectrometry (MS) is a routine approach for multiple analytes, including small molecules and peptides. Electrospray-based MS platforms are typically employed, as they provide highly reproducible outputs for batch processing of multiple samples. Quantitation using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry, while less commonly adopted, offers the ability to monitor analytes at significantly higher throughput and lower cost compared with ESI MS. Achieving accurate quantitation using this approach requires the development of appropriate sample preparation, spiking of appropriate internal standards, and acquisition to minimize spo