title: Fabrication of an on-line enzyme micro-reactor coupled to liquid chromatography-tandem mass spectrometry for the digestion of recombinant human erythropoietin.
abstract: Our aim was to develop a fast and efficient on-line method using micro-reactors for the digestion and deglycosylation of recombinant human erythropoietin extracted from equine plasma. The trypsin digestion micro reactors were fabricated using fused silica capillaries with either a dextran-modified coating or a porous monolith that was able to immobilise the enzyme. These were both found to be reasonably robust and durable, with the trypsin immobilised on dextran-modified fused silica capillaries offering better reproducibility than the micro-reactor based upon covalent attachment of this enzyme to the polymer. It is also evident that the enzyme attached micro reactors produced some tryptic peptides in a greater yield than in-solution digestion. A peptide-N-glycosidase F reactor was also fabricated and, when coupled with the trypsin reactor, the deaminated peptides T5 DAM and T9 DAM from recombinant human erythropoietin could also be detected by LC-ESI-MS/MS analysis. These results were better than those achieved using off-line digestion plus deglycosylation reactions and the analysis required far less time and effort to complete. The use of this on-line approach improved the sensitivity, efficiency and speed of our confirmation methodology that is based upon detecting the unique peptide segments of recombinant human erythropoietin that has been affinity extracted from positive equine plasma samples.
authors: Analytical & Environmental Science Division, School of Biomedical Sciences, King׳s College London, London SE1 9NH, UK; Singapore Turf Club Laboratory, 1 Turf Club Ave, Singapore Racecourse, Singapore 738078, Singapore. Electronic address: foohsiaoching@turfclub.com.sg.
authors: Hsiao Ching
authors: HC
authors: Foo
authors: Analytical & Environmental Science Division, School of Biomedical Sciences, King׳s College London, London SE1 9NH, UK.
authors: Norman W
authors: NW
authors: Smith
authors: Singapore Turf Club Laboratory, 1 Turf Club Ave, Singapore Racecourse, Singapore 738078, Singapore.
authors: Shawn M R
authors: SM
authors: Stanley
mesh_terms: D002853:Chromatography, Liquid; D003911:Dextrans / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000737:chemistry*; D006801:Humans; D020100:Hydrogels / Q000737:chemistry; D009862:Online Systems; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D003911:Dextrans; D004800:Enzymes, Immobilized; D020100:Hydrogels; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068817:Epoetin Alfa; D012822:Silicon Dioxide; D014357:Trypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
keywords: Dextran-modified; Immobilised enzyme microreactors; On-line digestion; Peptide-N-glycosidase F; Recombinant human erythropoietin