Solid-phase synthesis, isolation and analysis of a mouse protein, the macrophage migration inhibitory factor

Abstract

Murine macrophage migration inhibitory factor (mMIF) is an Mr 12 500 protein composed of all natural amino acid residues. Using the fluorenylmethoxycarbonyl chemistry for solid-phase peptide synthesis (SPPS) under special conditions, a stepwise approach was very successful leading to a crude product in unexpected high purity. After RP-HPLC isolation, a little mass difference of −12 u was determined for the received protein by matrix-assisted laser desorption ionization MS and ion spray MS and the failure sequence [Ser15]-mMIF identified by Edman degradation. The total solid-phase synthesis was repeated in the stepwise manner under the same conditions leading to the expected mMIF protein in high purity, which was confirmed by different analytical methods. Our results shows the reproducibility of our SPPS approaches to proteins and point out the importance of high-resolution mass spectrometry for a rapid and accurate analysis of such biopolymers.