Detection of synthetic protein isomers and conformers by electrospray mass spectrometry

Electrospray mass spectrometry (ESMS) has been used to investigate the structural properties of a protein prepared by total chemical synthesis. Construction of an analog of the tenth type III module from fibronectin (¹⁰F3) by chemical ligation of the unprotected synthetic peptides ¹⁰F3 (1-40) (α)COSH and BrAc(42-94)¹⁰F3 was found to give two major products, both of which possessed a mass corresponding to the expected product, [(COS)^40-41]¹⁰F3. Comparisons of the ESMS charge distributions obtained for these two synthetic products with that obtained for recombinant ¹⁰F3 suggested that one of the synthetic ¹⁰F3 analogs was correctly folded and the other was somehow misfolded. This was further confirmed by 1D and 2D NMR analysis. Exposure of the misfolded synthetic [(COS)^40-41]¹⁰F3 to high pH and elevated temperature followed by analysis using liquid chromatography-mass spectrometry revealed a β-ester linkage between residues Asn⁴² and Ser⁴³, 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 exchange rates of labile protons within the synthetic and recombinant ¹⁰F3s. This application, which allows the number of slow exchanging backbone amides within a protein to be calculated, revealed clear differences in the H-bonding networks of the folded and unfolded synthetic protein modules. Replacement of Ser⁴³ by an alanine was found to circumvent the N → O acyl shift, and the resulting synthetic protein analogue, [Ala⁴³, (COS)^40-41]¹⁰F3, possessed identical structural properties to recombinant ¹⁰F3.