Determination of the disulfide bond pattern of the endogenous and recombinant angiogenesis inhibitor endostatin by mass spectrometry.
Endostatin, a C-terminal fragment of collagen XVIII, is a promising protein drug which is in development for cancer therapy due to its anti-angiogenic activity. Although several endogenous molecular forms of human endostatin differing in their N-terminal length and their post-translational modifications (18.5-22 kDa) have been discovered, only one recombinant form of 20 kDa is used in clinical trials. This protein, recombinantly expressed in Pichia pastoris, contains four cysteines forming two disulfide bonds (Cys1-Cys4 and Cys2-Cys3). In contrast, there are conflicting data about the disulfide pattern of endogenous material. This report presents the disulfide analyses of both the endogenous circulating endostatins isolated from human hemofiltrate and the recombinant protein. The determination of the disulfide pattern was performed by Edman degradation, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization ion trap mass spectrometry (ESI-ITMS) performed in the off-line nanospray mode. All native and recombinant endostatins exhibited a Cys1-Cys4 (Cys(162)-Cys(302)) and Cys2-Cys3 (Cys(264)-Cys(294)) linkage. For a clear discussion of fragmented disulfide-bridged peptide chains obtained from MS(n) experiments, a modified general nomenclature is proposed.
John
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IPF PharmaCeuticals GmbH, Feodor-Lynen-Str. 31, D-30625 Hannover, Germany. H.John@IPF-PharmaCeuticals.de
Forssmann
W G
WG
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry*; D003094:Collagen / Q000737:chemistry*; D043170:Collagen Type XVIII; D004220:Disulfides / Q000737:chemistry*; D043169:Endostatins; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000970:Antineoplastic Agents; D043170:Collagen Type XVIII; D004220:Disulfides; D043169:Endostatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D010434:Pepsin A