DETECTION OF SYNTHETIC PROTEIN ISOMERS AND CONFORMERS BY ELECTROSPRAYMASS-SPECTROMETRY

Electrospray mass spectrometry (ESMS) has been used to investigate the structural properties of a protein prepared by total chemical synthes is. Construction of an analog of the tenth type III module from fibron ectin ((10)F3) by chemical ligation of the unprotected synthetic pepti des (10)F3 (1-40) (COSH)-C-alpha and BrAc(42-94)(10)F3 was found to gi ve two major products, both of which possessed a mass corresponding to the expected product, [(COS)(40-41)](10)F3. Comparisons of the ESMS c harge distributions obtained for these two synthetic products with tha t obtained for recombinant (10)F3 suggested that one of the synthetic (10)F3 analogs was correctly folded and the other was somehow misfolde d. This was further confirmed by 1D and 2D NMR analysis. Exposure of t he misfolded synthetic [(COS)(40-41)](10)F3 to high pH and elevated te mperature followed by analysis using liquid chromatography-mass spectr ometry revealed a beta-ester linkage between residues Asn(42) and Ser( 43), produced by an N --> O acyl shift rearrangement at Ser43, as the origin of the misfolding. ESMS was also used to measure the H-D exchan ge rates of labile protons within the synthetic and recombinant (10)F3 s. This application, which allows the number of slow exchanging backbo ne amides within a protein to be calculated, revealed clear difference s in the II-bonding networks of the folded and unfolded synthetic prot ein modules. Replacement of Ser43 by an alanine was found to circumven t the N --> O acyl shift, and the resulting synthetic protein analogue , [Ala(43), (COS)40-41](10)F3, possessed identical structural properti es to recombinant (10)F3. (C) 1995 Academic Press, Inc.