Endogenous Phosphopeptidomics Analysis by Using Mesoporous IMAC Materials

The analysis of endogenous phosphopeptides is crucial for identifying biomarkers of cellular signaling alterations and monitoring disease-related pathophysiological changes in vivo. While phosphoproteomics has seen remarkable progress in recent years, the field of endogenous phosphopeptidomics has lagged behind. A major challenge in endogenous phosphopeptidomics analysis lies in the presence of highly abundant nonmodified peptides and proteins within complex biological matrices, such as biofluids, which can obscure the detection of low-abundance phosphopeptides. In this chapter, we describe a protocol of a recently developed approach by utilizing Zirconium (IV)-grafted mesoporous beads for selective phosphopeptide enrichment, followed by high-resolution nanoRPLC-MS/MS analysis, enabling enhanced sensitivity and specificity in the detection of endogenous phosphopeptides from serum samples.