Optimized protocol for shotgun label-free proteomic analysis of pancreatic islets

Abstract

Pancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical-extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼ 1 µL and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the samplés characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of NOD mice exposed to low doses of fluoride ions (F-) and the primary pathways altered by the treatment.