Nano Flow LC-MS Analyses of Insulin Receptor Site-specific Phosphorylation Induced by Insulin Lispro and Basal Insulin Peglispro

摘要

Insulin receptor plays a central role in glucose homeostasis and is the key target for insulin drug discovery and development. Phosphorylation of insulin receptor is the first event in insulin signal transduction and determines the signaling pattern and strength. Monitoring phosphorylation kinetics is important to understand insulin-signaling regulation and develop effective insulin analogs with appropriate biology. Basal insulin peglispro (BIL), a 20kDa PEGylated version of insulin lispro, is a novel basal insulin in clinical testing with prolonged duration of action for treating diabetes. In this work, we performed a comprehensive comparison of site-specific insulin receptor phosphorylation with BIL and insulin lispro to investigate any potential effect of PEGylation on insulin receptor phosphorylation profile. To aid in quantitation, stable isotope labeling by amino acids in cell culture (SILAC) approach was used. HEK293 cells over-expressing human insulin receptor isoform B (hIR-B) were metabolically labeled with ~(13)C_6 ~(15)N_4-Arginine and ~(13)C)6 ~(15)N_2-Lysine and then treated with human insulin, which served as the internal standard. Unlabeled cells were treated with the insulin lispro or BIL. Labeled and unlabeled cells were mixed in the same amount. Insulin receptors were immunoprecipitated from cell lysate and treated with trypsin and Lys-C. The tryptic peptides were analyzed using a LTQ-Orbitrap Elite mass spectrometer coupled with a nano-LC. hIR-B phosphopeptides were identified using SQUEST search algorithm in Proteome Discoverer, and quantified from extracted ion chromatograms (XIC) of unlabeled and labeled phosphopeptides. Phosphorylation kinetics of 13 sites of the insulin receptor were analyzed, which included seven tyrosine residues, five serine residues, and one threonine residue. This analysis represents the largest number of insulin receptor phosphorylation sites assessed. In total, due to the multiplicity of phosphorylation on the peptides, 22 unique phosphopeptides were analyzed. Our results show that the phosphorylation pattern and kinetics of all the phosphopeptides induced by BIL and insulin lispro are essentially identical, indicating that PEGylation of insulin lispro does not affect the autophosphorylation response on hIR-B.