UNLABELLED: PURPOSE; The aim of this study was to investigate asparagine (Asn) deamidation and aspartate (Asp) isomerization and to measure the content of isoaspartate (isoAsp) in recombinant human interleukin-11 (rhIL-11). METHODS: The rhIL-11 control and heat stressed samples were characterized with trypsin and endoproteinase Asp-N peptide mapping, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI MS) and capillary electrophoresis (CE). The total isoAsp content and bioactivity were also assessed. RESULTS: Stress of rhIL11 at 30 degrees C for 6 weeks in liquid resulted in significant isomerization of Asp45 and Asp47. Isomerization of Asp51 and deamidation of Asn49 were also detected at low levels. The stressed rhIL-11 molecule contained 0.3 mol of isoAsp per mol of protein, compared to only 0.007 mol/mol of protein in the control. CONCLUSIONS: Asp and Asn residues, located in a loop structure of rhIL-11, undergo isoAsp formation under stressed conditions.
UNLABELLED: PURPOSE; The aim of this study was to investigate asparagine (Asn) deamidation and aspartate (Asp) isomerization and to measure the content of isoaspartate (isoAsp) in recombinant humaninterleukin-11 (rhIL-11). METHODS: The rhIL-11 control and heat stressed samples were characterized with trypsin and endoproteinase Asp-N peptide mapping, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry (ESI MS) and capillary electrophoresis (CE). The total isoAsp content and bioactivity were also assessed. RESULTS: Stress of rhIL11 at 30 degrees C for 6 weeks in liquid resulted in significant isomerization of Asp45 and Asp47. Isomerization of Asp51 and deamidation of Asn49 were also detected at low levels. The stressed rhIL-11 molecule contained 0.3 mol of isoAsp per mol of protein, compared to only 0.007 mol/mol of protein in the control. CONCLUSIONS:Asp and Asn residues, located in a loop structure of rhIL-11, undergo isoAsp formation under stressed conditions.
Authors: R J Harris; B Kabakoff; F D Macchi; F J Shen; M Kwong; J D Andya; S J Shire; N Bjork; K Totpal; A B Chen Journal: J Chromatogr B Biomed Sci Appl Date: 2001-03-10
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