| 12781465 |
Thevis M, Loo RR, Loo JA: Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds. J Am Soc Mass Spectrom. 2003 Jun;14(6):635-47.
Mass spectrometry, proteomics, and protein chemistry methods are used to characterize the cleavage products of 79 kDa transferrin proteins induced by iron-catalyzed oxidation, including a novel C-terminal polypeptide released upon disulfide reduction. Top-down electrospray ionization tandem mass spectrometry (ESI-MS/MS) of intact multiply-charged transferrin from a variety of species (human, bovine, rabbit, chicken) performed on a quadrupole time-of-flight mass spectrometer yields multiply-charged b (n)-products originating near residues 56-69 from the N-terminal region, in addition to their complementary y (n)-products. Incubation of transferrin with reductants, such as dithiothreitol (DTT) or tris (2-carboxyethyl)-phosphine (TCEP), yields an increase in multiple charging observed by ESI-MS and an increase in molecular weight consistent with disulfide reduction. However, mammalian transferrins release a 6-8 kDa fragment upon disulfide reduction. Protein acetylation and MS/MS sequencing demonstrate that the fragment originates from the C-terminus of the protein, and that it is a separate polypeptide linked via three disulfide bonds to the main transferrin chain. The existence of a separate C-terminal chain is not annotated in protein sequence databases and, to date, has not been reported in the literature. Iron-catalyzed cleavage induces fragments originating from both the N- and C-terminus of transferrin. |
37(0,1,1,7) |