<meta charset="utf8"></head><body>使用单吸头中的微珠阵列 （BIST） 进行基于凝集素的聚糖自动化分析

<ol>
	<li>Butler, M., Reichl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+cell+expression+systems.">Animal cell expression systems.</a> Adv Biochem Eng Biotechnol. 175, 1-36 (2021).</li><li>Silva, M. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+One+-+Lectin+biosensors+in+cancer+glycan+biomarker+detection.">Chapter One - Lectin biosensors in cancer glycan biomarker detection.</a> Adv Clin Chem. 93, 1-61 (2019).</li><li>Hiono, T., Nagai-Okatani, C., Kuno, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=4.07+-+Application+of+glycan-related+microarrays.">4.07 - Application of glycan-related microarrays.</a> Comprehensive Glycoscience (Second Edition). , 134-148 (2021).</li><li>Shimazaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin+bead+array+in+a+single+tip+facilitates+fully+automatic+glycoprotein+profiling).">Lectin bead array in a single tip facilitates fully automatic glycoprotein profiling).</a> Anal Chem. 91 (17), 11162-11169 (2019).</li><li>Shimazaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GlycoBIST:+A+System+for+automatic+glycan+profiling+of+a+target+protein+using+milli-bead+array+in+a+tip.">GlycoBIST: A System for automatic glycan profiling of a target protein using milli-bead array in a tip.</a> Curr Protoc Protein Sci. 99 (1), e103 (2020).</li><li>Mahmood, T., Yang, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blot:+Technique,+theory,+and+trouble+shooting.">Western blot: Technique, theory, and trouble shooting.</a> N Am J Med Sci. 4 (9), 429-434 (2012).</li><li>Bojar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+useful+guide+to+lectin+binding:+machine-learning+directed+annotation+of+57+unique+lectin+specificities.">A useful guide to lectin binding: machine-learning directed annotation of 57 unique lectin specificities.</a> ACS Chem Biol. 17 (11), 2993-3012 (2022).</li><li>Nagai-Okatani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2.0:+An+integrated+visualization+for+lectin+microarray-based+mouse+tissue+glycome+mapping+data+with+lectin+histochemistry.">2.0: An integrated visualization for lectin microarray-based mouse tissue glycome mapping data with lectin histochemistry.</a> J Proteome Res. 20 (4), 2069-2075 (2021).</li><li>Geisler, C., Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+glycoanalysis+with+Maackia+amurensis+lectins+requires+a+clear+understanding+of+their+binding+specificities.">Effective glycoanalysis with Maackia amurensis lectins requires a clear understanding of their binding specificities.</a> Glycobiology. 21 (8), 988-993 (2011).</li><li>Noro, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N+-glycan+structures+of+Wisteria+floribunda+agglutinin-positive+Mac2+binding+protein+in+the+serum+of+patients+with+liver+fibrosis.">N -glycan structures of Wisteria floribunda agglutinin-positive Mac2 binding protein in the serum of patients with liver fibrosis.</a> Glycobiology. 31 (10), 1268-1278 (2021).</li><li>Rawitch, A. B., Pollock, H. G., Yang, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroglobulin+glycosylation:+Location+and+nature+of+the+N-Linked+oligosaccharide+units+in+bovine+thyroglobulin.">Thyroglobulin glycosylation: Location and nature of the N-Linked oligosaccharide units in bovine thyroglobulin.</a> Arch Biochem Biophys. 300 (1), 271-279 (1993).</li><li>Dang, K., Zhang, W., Jiang, S., Lin, X., Qian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+lectin+microarrays+for+biomarker+discovery.">Application of lectin microarrays for biomarker discovery.</a> Chemistry Open. 9 (3), 285-300 (2020).</li><li>Chen, S., Qin, R., Mahal, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweet+systems:+Technologies+for+glycomic+analysis+and+their+integration+into+systems+biology.">Sweet systems: Technologies for glycomic analysis and their integration into systems biology.</a> Crit Rev Biochem Mol Biol. 56 (3), 301-320 (2021).</li><li>Dwivedi-Agnihotri, H., Srivastava, A., Shukla, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+biotinylation+of+purified+proteins+for+measuring+protein-protein+interactions.">Reversible biotinylation of purified proteins for measuring protein-protein interactions.</a> Methods Enzymol. 633, 281-294 (2020).</li><li>Shimazaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auto-lectin+dotcoding+by+two+octopuses:+Rapid+analysis+of+fluorescence-labeled+glycoproteins+by+an+8-channel+fully-automatic+bead+array+scanner+with+a+rolling-circle+detector.">Auto-lectin dotcoding by two octopuses: Rapid analysis of fluorescence-labeled glycoproteins by an 8-channel fully-automatic bead array scanner with a rolling-circle detector.</a> Anal Chem. 95 (32), 11868-11873 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL 8 PCR tubes + flat cap</td>
			<td>FastGene</td>
			<td>FG-0028FC/SE</td>
			<td>For dispensing and storage of biotin labeling reagents</td>
		</tr>
		<tr>
			<td>2.0 mL SC Micro Tube protein LB</td>
			<td>SARSTEDT AG&#38;Co. KG</td>
			<td>72.694.600</td>
			<td>For loading samples in LuBEA-VIII</td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>SAS Institute Inc.</td>
			<td>JMP 17</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Static Ionizer. 110 volts</td>
			<td>Plas-Labs</td>
			<td>800-AS/SPI</td>
			<td>Use while inserting beads into BIST Tip.</td>
		</tr>
		<tr>
			<td>Antistatic tweezers NK2A</td>
			<td>RUBIS&#160;</td>
			<td>9-5681-01</td>
			<td>Use while inserting beads into BIST Tip.</td>
		</tr>
		<tr>
			<td>Automated reaction measurement device</td>
			<td>Precision System Science Co., Ltd.</td>
			<td>LuBEA</td>
			<td>All data in this study were obtained using LuBEA-VIII. While LuBEA is commercialized, LuBEA-VIII is currently in the development phase. The website for product details of LuBEA and LuBEA-VIII can be found at the following URL (https://www.pss.co.jp/english/technology/apit/bist02.html).</td>
		</tr>
		<tr>
			<td>Biotin-(AC5)2 Sulfo-Osu</td>
			<td>Dojindo</td>
			<td>341-06801</td>
			<td>As biotinilation reagent</td>
		</tr>
		<tr>
			<td>BIST spacer bead- V-11</td>
			<td>Precision System Science Co., Ltd.</td>
			<td>F4938000</td>
			<td>Diameter: 1 mm</td>
		</tr>
		<tr>
			<td>BIST tip and sheath</td>
			<td>Precision System Science Co., Ltd.</td>
			<td>F4930-000</td>
			<td>Fill this empty tip with beads.</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Wako</td>
			<td>039-00475</td>
			<td>For preparing buffer solutions</td>
		</tr>
		<tr>
			<td>Can Get Signal Solution 2</td>
			<td>TOYOBO</td>
			<td>NKB-301</td>
			<td>This blocking reagent is good for this method</td>
		</tr>
		<tr>
			<td>CentriVap Benchtop Vacuum Concentrators</td>
			<td>Labconco</td>
			<td>7810010</td>
			<td>For dry of biotinilation reagent. The substitution provided by other companies is possible.</td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate&#160;</td>
			<td>Bio-Rad</td>
			<td>1705060</td>
			<td>For measuring the samples by LuVEA VIII</td>
		</tr>
		<tr>
			<td>Cold trap CentriVap series</td>
			<td>Labconco</td>
			<td>7460040</td>
			<td>For dry of biotinilation reagent. The substitution provided by other companies is possible.</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Wako</td>
			<td>077-00735</td>
			<td>For preparing buffer solutions</td>
		</tr>
		<tr>
			<td>Manganese(II) Chloride Tetrahydrate</td>
			<td>Wako</td>
			<td>139-00722</td>
			<td>For preparing buffer solutions</td>
		</tr>
		<tr>
			<td>Milli-Q reference</td>
			<td>MERCK</td>
			<td>ZIQ7000T0C</td>
			<td>As deionazed water for dilution of reagents and buffers</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Wako</td>
			<td>162-19321</td>
			<td>For preparing buffer solutions</td>
		</tr>
		<tr>
			<td>Peroxidase Streptavidin</td>
			<td>Jackson</td>
			<td>016-030-084</td>
			<td>This antibody is good for GlycoBIST analysis. It is useful for the detection of the western blotting, although substitutions can be provided by other companies.</td>
		</tr>
		<tr>
			<td>Probe bead</td>
			<td>Precision System Science Co., Ltd.</td>
			<td>FP4936000</td>
			<td>For preparing lectin-fixed beads. See Shimazaki, H. et al., Current Protocols in Protein Science, 2020 in reference section for detail.</td>
		</tr>
		<tr>
			<td>Protein LoBind Tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>0030108442</td>
			<td>Use while preparing the samples such as biotinylation.</td>
		</tr>
		<tr>
			<td>Silica gel (for desiccan)</td>
			<td>Kanto chemical</td>
			<td>37039-02</td>
			<td>Use while storing the GlycoBIST tip and dried biotinylation reagent.</td>
		</tr>
		<tr>
			<td>Static electricity removal sheet M</td>
			<td>TRUSCO NAKAYAMA</td>
			<td>SD5050</td>
			<td>Use while inserting beads into BIST Tip.</td>
		</tr>
		<tr>
			<td>SV Cartridge II</td>
			<td>Daido Chemical Industry</td>
			<td>ED041</td>
			<td>For measuring the samples by LuVEA VIII</td>
		</tr>
		<tr>
			<td>Table Microcentrifuge-CAPSULEFUGE</td>
			<td>TOMY</td>
			<td>PMC-060</td>
			<td>For spin-down of the mixture in each process</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai tesque</td>
			<td>35501-15</td>
			<td>For preparing buffer solutions</td>
		</tr>
	</tbody>
</table>

rapid glyco-qualitative assessment of recombinant proteins using a fully automated system

Protein glycosylation is a crucial post-translational modification that needs to be assessed in biopharmaceuticals. The glycan profiles of proteins can vary based on culture conditions, purification processes, and host cells1. Simple instruments are required to qualify glycosylation within the bioprocess pipeline. It is estimated that over 50% of secreted and membrane proteins in vivo are modified with multiple glycans, which change according to cell lineage, developmental stage, and disease status, such as the onset of malignancy2. Monitoring glycan profiles holds significant potential for identifying unique diagnostic markers and drug targets. Automated instruments capable of rapidly measuring large-scale sample sizes are in high demand to verify and validate such aberrant glycosylation from hundreds of patient samples in the discovery pipeline.
Microarray technology has been incorporated into glycomics for evaluating the glycan profiling of glycoproteins3. In this method, several lectins, which are glycan-binding proteins, are immobilized on a surface such as a glass slide. This interaction-based glyco-analysis technology does not necessitate releasing glycans from core proteins beforehand, simplifying the process for researchers new to glycotechnology. Despite its widespread use, for industrial applications such as bioproduction, an automated system capable of quickly and easily monitoring glycans for a larger number of analysis targets was required. To address this, an automated glycan profiling system based on a unique concept termed &#34;bead array in a single tip&#34; (BIST), initially developed for genotyping, was previously reported. This system simplifies the process with a one-box type high-throughput auto-instrument4. Using tips in which various lectin-fixed beads are arranged in parallel4,5, a method for analyzing glycan structures modified in glycoproteins was established and named GlycoBIST (hereafter referred to as &#34;automatic glycan profiling system&#34;) (Figure 1A). Lectins can be fixed on 1,000 beads and dried to maintain activity for a year, both before and after packing in a tip. Once tips and cartridges containing reagents such as HRP-labeled anti-streptavidin antibody (SA-HRP) are set in the measuring prototype instrument (an automated reaction measurement device, see Table of Materials), the tip functions as an autopipette. A chemiluminescence detection scanner at the instrument&#39;s interior rear quantifies the signals of eight tips simultaneously. The quantitative data from these eight tips are compactly and concurrently displayed as dot codes on the instrument&#39;s touchscreen for quick confirmation of measurement results. Furthermore, the value represented as the maximum of the measured peak is transported from the instrument as raw data, and it allows for graphing by individual researchers (Figure 1A, bottom panel).
In this article, the authors describe an improved method of biotin-labeling proteins, reducing processing time to 30 min. Target proteins are biotinylated in advance and detected by SA-HRP (Figure 1B). A standard GlycoBIST tip (specialized tip for automatic glycan profiling) with 15 selected lectins was constructed to achieve the versatile glycoprotein glycan profiling for analytical comprehensiveness.

<meta charset="utf8"></head><body>作者聚焦：使用全自动系统推进蛋白质糖基化研究

使用全自动系统对重组蛋白进行快速糖质定性评估

Our scope is protein glycosylation. Glycan structures are involved in changes in the stability and function of biopharmaceuticals and are altered along with host cell species and culture conditions. In some production conditions, allergenic glycan would be contaminated to a certain degree. Our goal is to make user-friendly tools for such glyco-qualification.Mostly, LC and Mass spectrometry. But, for simple testing, lectin-based technologies are applicable. Researchers have available many types of lectins with different specificities and each lectin binds to some specific glycans. So, we can estimate glycan structures using multiuse of lectin-by-lectin blotting, lectin ELISA, and lectin microarray.Glycosylation occurs in functional proteins in our body. Glycan structure is altered along with diseases. Nowadays, we have used several disease-specific glycosylations as diagnostic biomarkers, such as CA 19-9 and AFP-L3. Notably, new biomarker candidates have been discovered using lectin-based technologies. For their validation, we need glyco-qualification system.Glyco-qualification is hard work. We need to analyze hundreds or thousands of numbers in a short time. However, there is no system to meet the needs. Our analytical method is the only one with a full-automatic measurement process. It drastically reduces manpower and enables us to conduct effective validation.Our work aims to further improve our rapid instrument, especially for quantitative measurement. After this, we expect to realize a unique diagnostic tool with "glycobiomarker panel" measured by our multilectin system. Hence, we are focusing on the discovery of disease-specific glycobiomarkers.

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

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

Research

JoVE Journal

Biochemistry

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

489 Views.  National Institute of Advanced Industrial Science and Technology. Our scope is protein glycosylation. Glycan structures are involved in changes in the stability and function of biopharmaceuticals and are altered along with host cell species and culture conditions. In some production conditions, allergenic glycan would be contaminated to a certain degree. Our goal is to make user-friendly tools for such glyco-qualification.Mostly, LC and Mass spectrometry. But, for simple testing, lectin-based technologies are applicable. Researchers have available ma...