Fourier-transform mass spectrometry for detection of thioester-bound intermediates in unfractionated proteolytic mixtures of 80 and 191 kDa portions of Bacitracin A synthetase Original Research Article

Enzymes involved in polyketide and non-ribosomal peptide biosynthesis utilize covalent intermediates on several carrier sites along their exceptionally large primary structures (100–1000 kDa). Direct detection of these thioester-bound intermediates using mass spectrometry (MS) could dissect the timing of intermediate formation and translocation. Proteolysis of BacA1 (80 kDa), the first module in the production of Bacitracin A, using Endoproteinase Glu-C yielded 202 peptides from 1 to 20 kDa as detected using electrospray ionization with Fourier-transform MS at 9.4 T. A 1:1 mixture of Ile and 13C6-Ile allowed the selective detection of the Ile-thioester bound intermediate directly from the 200-component mixture. Direct FTMS analysis of the Glu-C digestion products from the two module BacA1-2 construct (191 kDa) gave perhaps the most complicated spectrum of a proteolytic digest ever recorded at isotopic resolution. This 9.4 T spectrum (1000 scans) contains 759 individual isotopic distributions from 528 peptides from 1 to 30 kDa. While the Glu-C product from the BacA1 carrier site was again detected, a peptide corresponding to the second carrier site was not. This indicates that even using the highest resolution type of mass spectrometry, some peptide fractionation (e.g., using capillary chromatography) will be required to detect thioester-bound intermediates in a robust fashion from such large “assembly line” catalysts.