Protein glycosylation is acknowledged to be one of the major post-translational modifications, which significantly affects protein folding, conformation, stability and activity. Glycosylation profiling is a key issue in the production of biotherapeutics and biosimilars as well as in the identification of circulating biomarkers for several pathologies. Notwithstanding the continuous progress in the field, the basic pipeline for glycosylation profiling is still time consuming and analytically challenging. In this context, higher automation and throughput can be achieved by developing tailored analytical tools, such as immobilized enzyme reactors (IMERs), to be used as alternative to time- and material-consuming in solution enzymatic reactions. This presentation focuses on the development of a novel IMER containing Peptide-N-glycosidase F (PNGase F), one of the most frequently used endoglycosidases in glycomics, its characterization and its integration into a LC-ESI-Q-ToF platform, to enable the sequential analysis of both deglycosilated proteins and released glycans. The PNGase F-IMER was obtained by oriented covalent immobilization of the target enzyme onto an epoxy CIMac™ analytical column. The obtained IMER was characterized and inserted into an integrated LC-MS platform. In the optimized set up, glycoproteins were first on-line deglycosylated by the PNGaseF-IMER, the deglycolysalted proteins were subsequently collected by a C4 CIMac™ analytical column, and analysed by ESI-Q-ToF. In parallel, released glycans were trapped onto a Hypercarb porous graphitic carbon column and sent to ESI-Q-ToF for analysis and identification. Each step was optimized to be perfectly interfaced with the subsequent MS analysis as well as with the enzyme requirements for stability and activity.

Developments of an integrated PNGase-F-IMER-LC-ESI-Q-ToF platform for glycoprotein analysis

BARTOLINI, MANUELA
2016

Abstract

Protein glycosylation is acknowledged to be one of the major post-translational modifications, which significantly affects protein folding, conformation, stability and activity. Glycosylation profiling is a key issue in the production of biotherapeutics and biosimilars as well as in the identification of circulating biomarkers for several pathologies. Notwithstanding the continuous progress in the field, the basic pipeline for glycosylation profiling is still time consuming and analytically challenging. In this context, higher automation and throughput can be achieved by developing tailored analytical tools, such as immobilized enzyme reactors (IMERs), to be used as alternative to time- and material-consuming in solution enzymatic reactions. This presentation focuses on the development of a novel IMER containing Peptide-N-glycosidase F (PNGase F), one of the most frequently used endoglycosidases in glycomics, its characterization and its integration into a LC-ESI-Q-ToF platform, to enable the sequential analysis of both deglycosilated proteins and released glycans. The PNGase F-IMER was obtained by oriented covalent immobilization of the target enzyme onto an epoxy CIMac™ analytical column. The obtained IMER was characterized and inserted into an integrated LC-MS platform. In the optimized set up, glycoproteins were first on-line deglycosylated by the PNGaseF-IMER, the deglycolysalted proteins were subsequently collected by a C4 CIMac™ analytical column, and analysed by ESI-Q-ToF. In parallel, released glycans were trapped onto a Hypercarb porous graphitic carbon column and sent to ESI-Q-ToF for analysis and identification. Each step was optimized to be perfectly interfaced with the subsequent MS analysis as well as with the enzyme requirements for stability and activity.
2016
7th Monolith Summer School and Symposium (MSS2016) Abstract Book
L10
L10
Bartolini, Manuela
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/598910
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