title: Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095-1570, USA.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.forename: Rachel R Ogorzalek
authors.initials: RR
authors.lastname: Loo
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002645:Chickens; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007501:Iron / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011817:Rabbits; D021241:Spectrometry, Mass, Electrospray Ionization; D013439:Sulfhydryl Reagents / Q000737:chemistry; D014168:Transferrin / Q000737:chemistry*
chemical_list: D004220:Disulfides; D010446:Peptide Fragments; D010455:Peptides; D013439:Sulfhydryl Reagents; D014168:Transferrin; D007501:Iron; D004229:Dithiothreitol