Biotage : Mass Directed Flash Purification of Amphipathic Peptide ‘18A’

The use of fast and precise microwave heating in solid phase peptide synthesis can result in peptides of greater purity, thereby simplifying the subsequent purification steps. In this application note, we demonstrate that reversed-phase mass directed flash purification can be used to separate deletion peptides with closely related sequences.

Mass-based fractionation offers the advantage of being able to separate products with no UV signal, and also resolve peaks with overlapping UV signals, e.g. where incomplete incorporation of amino acids during peptide synthesis leads to deletion sequences. These peptide side-products have similar physicochemical properties and tend to elute in similar conditions to the intended product and thus repre¬sent a major purification challenge.

The amphipathic peptide called '18A' has been used for structural studies on lipid-peptide particles which form peptide nanodiscs and are a possible solution for the handling of membrane proteins. Here we synthesized two closely related amphipathic peptides and show the mass directed flash purification of non-acetylated '18A' from a crude mixture containing a single residue deletion sequence '17A' (-Glu residue) using the Biotage® Isolera Dalton™ mass-directed flash chromatography system.

Peptide '18A' forms nanodiscs that can hold transmembrane proteins.

We demonstrate that reversed-phase mass directed flash purification can be used to separate deletion peptides with closely related sequences. Fractionation based on compound mass was able to identify the peptides of interest. Mass directed fractionation using reversed-phase flash chromatography is a very effective purification technique in terms of cost efficiency and speed (by reducing solvent use and time) and can provide sufficiently pure materials that can be used for downstream applications. Thus using the Biotage® Initiator+ Alstra™ and Biotage® Isolera Dalton™ for the synthesis and purification of peptides is a powerful combination to improve workflow from crude peptide to purified product.