Determination of the limited trypsinolysis pathways of tumor necrosis factor-α and its mutant by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-a (wtTNF-α) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-α by trypsin occurs sequentially: Arg², Arg⁶, Arg³², Arg³¹, and Arg⁴⁴, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-α and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.