| Autor: |
Yang, Thao, Horejsh, Douglas R., Mahan, Kermit J., Zaluzec, Eugene J., Watson, Throck J., Gage, Douglas A. |
| Zdroj: |
Analytical Biochemistry; November 1996, Vol. 242 Issue: 1 p55-63, 9p |
| Abstrakt: |
The combined use of trypsin digestion and peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported here as an effective and rapid means for identifying the cross-linking sites in human oxy hemoglobin A (HbA) cross-linked with either bis(3,5-dibromosalicyl)-succinate or -glutarate. MALDI-MS analysis of a nondigested sample of oxy HbA modified with bis(3,5-dibromosalicyl)-glutarate showed that cross-linking only occurred between the β1- and β2-protomers and not between α1- and α2- or α- and β-protomers, along with a modification reaction on an un-cross-linked β-chain. Results of the MALDI tryptic peptide mass maps of cross-linked hemoglobins showed several cross-linked peptides having masses consistent with: βVal67-Lys95-XL-βVal67-Lys95, βVal67-Lys95-XL-βVal67-Arg104, βVal67-Arg104-XL-βVal67-Arg104, where XL represents the succinyl or glutaryl bridging span moiety. Each of these peptides contains Lys82, the targeted residue for these reagents, substantiating the cross-linking sites at β1Lys82-β2Lys82. This approach in general will enable rapid identification of the cross-linking sites in engineered proteins or intracellularly recombinant cross-linked proteins when the mass of the cross-linker and the protein primary structure are known. |
| Databáze: |
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