Mass spectrometry MS analysis and selective enrichment

Mass spectrometry (MS) analysis and selective enrichment methods of phosphorylated proteins are powerful tools that can help address these challenges. Recent advances in MS have made it possible to analyze signaling pathways by facilitating high-throughput identification of phosphorylation sites with high accuracy and sensitivity [9], [10]. Furthermore, the revolutionary development of post-translational modification (PTM) enrichment methods has enabled the identification and quantification of PTMs. Specific enrichment methods are essential to detect components in low abundance in complex mixtures. Several enrichment methods specific to phosphorylated peptides have been developed [e.g., immobilized metal affinity chromatography and immunoprecipitation using phospho-specific antibodies] [11]. Ishihama et al. [12] recently developed a highly selective method for isolating phosphorylated peptides using hydroxyl acid-modified metal oxide chromatography (HAMMOC). In the present study, we identified precise phosphorylation sites of Adrb1 in vivo. Previous studies provided detailed mapping data of ADRB2 phosphorylation sites and extensively studied their site-specific functions [5], [13]. Although putative phosphorylation sites of human ADRB1 have been identified using site-directed mutagenesis experiments in cell lines [14], [15], there have been no reports on Adrb1 phosphorylation sites in mammalian tissues. Therefore, we used phosphoproteomics techniques to precisely identify phosphorylation sites of Adrb1 in the mouse heart.
Materials and methods
Results
Discussion Previous studies have demonstrated that phosphorylated residues in the C-terminus of ADRB2 are required for cardiac desensitization and DLPC synthesis [13]. In contrast, the functional significance of phosphorylated residues in Adrb1 is less well-characterized. Serine 312 in the third intracellular loop of human ADRB1 is the only ADRB1 residue reported to be a phosphorylation substrate of protein kinase A, and it has been shown to play a critical role in recycling and functional resensitization in the cell model [14]. Several putative ADRB1 phosphorylation sites have been investigated [15]; however, these sites have yet to be confirmed, even in cell lines. Studies designed to map phosphorylation sites in GPCRs and investigate the functional significance of these sites have primarily been conducted in cell lines [3]. Although these studies can help identify the distinct biological function of phosphorylated residues, there might be significant differences in the regulation of phosphorylation events between in vivo and in vitro conditions. Therefore, accurate mapping results are typically derived from a combination of in vivo and in vitro data. To comprehensively characterize the function of distinct phosphorylation events, it is necessary to identify phosphorylation sites in vivo under physiological conditions. In this context, MS is a powerful tool for identifying and quantifying individual phosphorylation sites with high sensitivity, selectivity, and throughput using only a small quantity of analyte [9], [10]. Nonetheless, a “more is better” rule would be applied to the amount of starting material required for the successful MS analysis. On the contrary, isolating large amounts of protein from tissues can often be difficult. Furthermore, the stoichiometric level of phosphorylated protein is extremely low compared with the unmodified protein. Therefore, it is necessary to get enough amount of protein to acquire high sequence coverage and obtain high quality MS spectra. In addition, to reduce the complexity of sample, it is necessary to conduct enrichment procedures or to fractionate the sample at the protein or peptide level. Using an enrichment- and fractionation-based approach, we identified four phosphorylated peptide fragments in the C-terminus and third cytoplasmic loop of Adrb1 from crude protein membrane extracts of four mouse hearts (Table 1). However, no Adrb1 fragments were detected in an analysis of total proteins derived from a single intact heart (data not shown), indicating the effectiveness of this strategy.