Development and Application of Phosphoproteomic Approaches for Quantitative Protein Phosphorylation Analysis and Global Phosphoproteome Profiling Using Mass Spectrometry

Abstract

Phosphorylation plays a pivotal role in functionalities of proteins, including their activity, subcellular localization, molecular interactions and stability. This reversible mechanism modulates many cellular processes, as phosphorylation is molecular switch based on the temporal and spatial balance of kinases and phosphatases. In particular, phosphorylation is responsible for cellular transduction signaling and abnormal phosphorylation contributes to the onset and progression of various diseases. Consequently, research of individual protein phosphorylation and global phosphoproteome can provide disease-associated signatures as well as relevant information on cellular processes. Recent advances over the last several years in mass spectrometry enhance the coverage of individual protein and global phosphoproteome profiling but analytical challenges remain in sample preparation, phosphopeptide enrichment, and computational proteomics. Therefore, I have developed and applied phosphoproteomic approaches for quantitative targeted protein phosphorylation analysis and global phosphoproteome profiling using mass spectrometry. In part I, I developed quantitative phosphorylation analysis method to monitor the phosphorylation changes of transgelin2 under different kinase activation conditions. Transgelin2, one of cytoskeletal actin binding proteins has recently been suggested to be involved in the formation of immune synapses. Although detailed function of transgelin2 is largely unknown, interactions between transgelin2 and actin appear to be important in regulating cellular functions of transgelin2. Because protein phosphorylation can change ability to interact with other proteins, comprehensive phosphorylation analysis of transgelin2 will be helpful in understanding its functional mechanisms. Thus, I employed a specific protein label-free quantitative phosphorylation analysis method combining immuno-precipitation, immobilized metal ion affinity chromatography (IMAC) phosphopeptide enrichment technique and label-free relative quantification analysis to monitor the phosphorylation changes of transgelin2 overexpressed in Jurkat T cells under protein kinase C (PKC) and protein kinase A (PKA) activation conditions, two representative intracellular signaling pathways of immune cell activation and homeostasis. Six serine/threonine phosphorylation sites were identified including threonine-84, a novel phosphorylation site. Notably, distinct phosphorylation patterns of transgelin2 under the two kinase activation conditions were observed. Most phosphorylation sites showing specific kinase-dependent phosphorylation changes were discretely located in two previously characterized actin-binding regions: actin-binding site (ABS) and calponin repeat domain (CNR). Accordingly, this study data suggest that different actin-binding properties or cellular functions of transgelin2 may result from distinct intracellular signaling events under immune response activation or homeostasis conditions. In part Ⅱ, I optimized serial phosphopeptide enrichment methods for comprehensive plasma phosphoproteome profiling. The plasma phosphoproteome, like the plasma proteome, is a valuable noninvasive and economical biomarker source for various diseases. Nonetheless, the discovery and verification of plasma phosphoprotein biomarkers have been hampered by the inherent characteristics of plasma that are detrimental for phosphopeptide enrichment. Here, I expanded the coverage of the plasma phosphoproteome by optimizing existing serial phosphopeptide enrichment methods. This method identified 2,297 phosphopeptides from plasma samples of healthy individuals. Notably, plasma phosphoproteome in this study showed the strongest functional association with the phosphoproteome from brain rather than from other blood components or major organs. Importantly, Alzheimer’s disease (AD)-related phosphorylated brain proteins, including Tau, were detected in this plasma phosphoproteome. I thus demonstrate that AD-related brain proteins in the plasma phosphoproteome can be used as a biomarker for AD diagnosis by targeted mass spectrometry analysis of plasma samples from patients at normal or preclinical stages, and those with mild cognitive impairment and dementia. Targeted phosphopeptides showed progressive increases from normal− to normal+, MCI+, and AD+, indicating their diagnostic power for AD. Therefore, plasma phosphoproteome analysis in this study represents a potentially powerful noninvasive tool for early diagnosis of AD and further distinguishes MCI+ and AD+ patients.