Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds.
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.
Thevis
Mario
M
Department of Chemistry and Biochemistry, University of California at Los Angeles, Los Angeles, California 90095-1570, USA.
Loo
Rachel R Ogorzalek
RR
Loo
Joseph A
JA
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*
D004220:Disulfides; D010446:Peptide Fragments; D010455:Peptides; D013439:Sulfhydryl Reagents; D014168:Transferrin; D007501:Iron; D004229:Dithiothreitol