O- and N-Phosphoproteomic Analysis Using Online Alkaline-pH Reversed-Phase Nanoelectrospray-Tandem Mass Spectrometry

Protein phosphorylation (O-linked and N-linked) is associated with a wide range of biological processes and cell signaling pathways. Aberrant phosphorylation is often observed as a hallmark of diseases, including cancer, neurodegenerative disorders, and metabolic syndromes. Mass spectrometry (MS) is an indispensable tool for studying protein phosphorylation. This is primarily due to its high sensitivity and throughput, which allows for comprehensive identification and quantification of phosphorylation events in complex biological samples. In this chapter, we describe a detailed protocol for phosphoproteomic analysis using one-dimensional online alkaline-pH reversed-phase nanoelectrospray-tandem MS (alkaline-pH-MS/MS). It complements traditional online low-pH reversed-phase nanoelectrospray-tandem MS (low-pH-MS/MS) by modulating the charge state distribution of phosphopeptides, which may facilitate the characterization of phosphoproteins with significant biological functions.