rapid-glyco-qualitative-assessment-recombinant-proteins-using-fully

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Protein glycosylation, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, ...

National Institute of Advanced Industrial Science and Technology

Precision System Science Co., Ltd.

Wisteria floribunda agglutinin positive human Mac-2-binding protein (WFA(+)-hM2BP) was recently validated as a liver fibrosis glycobiomarker with a fully automated lectin-antibody sandwich immunoassay. In this study, we supplied recombinant WFA(+)-hM2BP as the standard glycoprotein and the overlaid antibody to enhance the robustness of WFA(+)-hM2BP quantification.

Reconstruction of a robust glycodiagnostic agent supported by multiple lectin-assisted glycan profiling.

Nearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker.

Alteration of matrix metalloproteinase-3 O-glycan structure as a biomarker for disease activity of rheumatoid arthritis.

A quantitative description of glyco-alteration/differences in diseases can lead to the development of a diagnostic agent for use in vitro to monitor the degree of change in target glycoproteins. Analytical systems have been developed along with the progress of omics-oriented technologies. For clinical implementation, their full automation is required with an apparatus that is simple to operate. Here, we report an automatic analysis system for quantitative characterization of glyco-alteration/differences that depends on the unique strategy of "bead arrays in a single tip." The alternative lectin array can obtain a minimum characterization of the glycan profile for nanogram quantities of an endogenous glycoprotein. A simple autopipetting robot produces the precise chemiluminescence detection of glycan-lectin interactions with a wide dynamic range that is superior to fluorescence-based lectin arrays. The tip-based array format enables automatic glycan profiling from sample pretreatment to detection with low variation and linear detection, which may facilitate the use of this lectin array in clinical practice.

Lectin Bead Array in a Single Tip Facilitates Fully Automatic Glycoprotein Profiling.

Lectin is a biomolecule that recognizes a specific part of glycans and, thus, has been used widely as a probe for glycoprotein analysis. Owing to the wide repertoire in nature combined with the recent two decades of advances in microarray technology, the multiplexed use of lectins has been widely used for glycan profiling of endogenous proteins. Because protein glycosylation is recognized as being biologically important and is expected to be a reliable disease marker, lectin microarray analysis with highly sensitive detection has been used to discover disease-relevant glycosylation alterations. However, the conventional system is limited to research purposes; thus, its implementation in clinical settings is warranted. Here, we provide an automatic glycan profiling method using GlycoBIST. A unique array format is used for 10-plexed lectin-glycoprotein interaction analysis on 1-mm-sized beads, which are arranged vertically in a capillary-shaped plastic tip. Using a one-boxed autopipetting machine, the whole process (including interaction, washing, and detection) is performed automatically and serially, resulting in reproducible measurements. In this article, a typical method for glycan profiling of a purified glycoprotein and the fabrication of GlycoBIST tips is explained. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Fabrication of a GlycoBIST tip Basic Protocol 2: Automatic profiling of a target glycoprotein using GlycoBIST.

Hiroko Shimazaki

Molecular & Cellular Glycoproteomics Research Group,
Cellular and Molecular Biotechnology Research Institute,
Molecular & Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute

National Institute of Advanced Industrial Science and Technology

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Hiroko Shimazaki

Molecular & Cellular Glycoproteomics Research Group,Cellular and Molecular Biotechnology Research Institute,Molecular & Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute

National Institute of Advanced Industrial Science and Technology

Rapid Glyco-Qualitative Assessment of Recombinant Proteins Using a Fully Automated System

Molecular & Cellular Glycoproteomics Research Group,
Cellular and Molecular Biotechnology Research Institute,
Molecular & Cellular Glycoproteomics Research Group, Cellular and Molecular Biotechnology Research Institute