Membrane Protein Profiling of Human Islets of Langerhans Using Several Extraction Methods

摘要

Introduction Proteomic characterization of the human pancreatic islets, containing the insulin producing beta-cells, is likely to be of great importance for improved treatment and understanding of the pathophysiology of diabetes mellitus. Objective The focus of this study was to characterize the human islet membrane proteome.Methods In order to identify as many membrane proteins as possible, five different extraction procedures were used, i.e., phase separation using Triton X-114, a plasma membrane protein kit, cell surface protein biotinylation, total protein extraction, and lipid-based protein immobilization flow cell. Digested protein extracts were analyzed by nanoflow liquid chromatography tandem mass spectrometry. Then the identified proteins were categorized according to cellular location using their gene ontology annotation and by prediction of transmembrane helices in the sequence. This information was used to estimate the amount of membrane proteins identified. Results By combining the results from all extraction procedures, the total number of membrane proteins identified from the human islets was increased, accentuating that a combination of methods usually gives a higher coverage of the proteome. A total of 1,700 proteins were identified (≥2 unique peptides), and 735 of these proteins were annotated as membrane proteins while 360 proteins had at least one predicted transmembrane helix. The extraction method using phase separation with Triton X-114 yielded both the highest number and the highest proportion of membrane proteins. Conclusion This study gave an enhanced characterization of the human islet membrane proteome which may contribute to a better understanding of islet biology.