Monitoring protein cleavage and concurrent disulfide bond assignment using thermospray LC/MS

The arrangement of the disulfide bridges of Cucurbita maxima trypsin inhibitor, CMTI I, has been confirmed by enzymatic and chemical cleavages of the native protein and analysis of the resulting disulfide-bridged fragments using thermospray liquid chromatography/mass spectrometry. Although the disulfide bridges of CMTI I have recently been assigned from the x-ray crystallographic structure, direct chemical analysis of the S-S bonds using classical techniques proved difficult. The CMTI I molecule is extremely resistant to enzymatic digestion, and only one site of the peptide chain (Met-8) can be used efficiently for chemical cleavage. A series of degradative conditions were employed in the studies reported here. The progress of protein modification was monitored directly by high-performance liquid chromatography/thermospray mass spectrometry. The disulfide pairings could be deduced directly from the mass spectra of the peptides produced by the fragmentation processes and resolved by high-performance liquid chromatography. In two instances, fragments involving a disulfide bond were isolated and further analyzed, and these confirmed the mass spectral assignments. The disulfide bridges identified, 3-20, 10-22 and 16-28, correspond to those of the x-ray structure and are consistent with those assigned for two other closely related trypsin inhibitors.