Title: The Concept of Functional Peptidomics for the Discovery of Bioactive Peptides in Cell Culture Models
Volume: 8
Issue: 8
Author(s): Marco M. Jost, Petra Budde, Harald Tammen, Rudiger Hess, Markus Kellmann, Imke Schulte and Horst Rose
Affiliation:
Keywords:
Adipocytes, Bioactivity, Differential Peptide Display, Functional Peptidomics, Glucose uptake, INS-1, Lipolysis, Proinsulin processing, Reversed-phase HPLC
Abstract: Detection and purification of novel bioactive peptides from biological sources is a scientific task that led to a substantial number of important discoveries. One major laborious approach used is the repetitive stepwise separation of the test sample into several fractions followed by the determination of their bioactivity, until purity allows for sequence identification. We tested whether functional peptidomics, a combination of biological read-outs with differential peptide display (DPD) is a suitable strategy to isolate bioactive peptides at lower workload and with improved success. Additionally, we evaluated the use of DPD to monitor the processing status of proinsulin by inhibition of the insulin processing pathway. The rat insulinoma cell line INS-1 stimulated either with 2 mmol/l or 10 mmol/l glucose was used as model to generate differential peptide displays. In parallel, the bioactivity of the supernatants from the INS-1 cells was measured by glucose uptake and lipolysis assays using the adipocyte cell line 3T3-L1. We were able to quickly and elegantly trace the known activity of insulin to increase glucose uptake and inhibit lipolysis. Following re-chromatography of selected fractions, relevant peptides were identified by DPD and bioassays: the rat insulin-1 precursor and two different insulin peptides. We demonstrated in a semi-quantitative fashion that inhibition of proinsulin processing leads to accumulation of the insulin precursor, and reduced secretion of insulin-1. Thus, we conclude that DPD is an attractive support technology in peptide purification strategies aiming to identify bioactive compounds, and is superior to ELISA in discriminating between the processing status of insulin and its precursor.
