This work was supported by the Adaptable and Seamless Technology transfer Program through Target-driven R&#38;D (A-STEP), funded by the Japan Science and Technology Agency (JST), under Grant Number JPMJTR204A and in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 23H02680 to AK.We would like to thank Editage (www.editage.jp) for editing the English language.

The details of the reagents and the equipment used for this study are listed in the Table of Materials.
1. Preparation of the specialized tip for automatic glycan profiling
<ol>
	<li>Bring the dried lectin-fixed beads, stored at 4 &#176;C in a sealed bag, to the bench and allow them to return to room temperature (~23 &#176;C). 
	NOTE: This prevents condensation on the beads, which can diminish their quality. This method of preparing lectin-fixing beads has been previously reported5.</li>
	<li>Place an antistatic cloth on the desk and place the portable static eliminator facing the workspace. This allows subsequent work to be performed on the bench with antistatic protection (Figure 2A).</li>
	<li>Insert beads into the empty tip using antistatic tweezers such that two spacer beads are placed between the lectin-fixing beads (Figure 1A).</li>
	<li>Gently squeeze the top of the tip with nippers to close the opening, being careful not to cut the tip after all beads have been filled (Figure 2B). 
	NOTE: This is to secure the beads inside the tip. It is recommended that the nipper be crafted to avoid cutting the tip (Figure 2C).</li>
	<li>Straighten the tip by hand if it is bent.</li>
	<li>Store the prepared tip at 4 &#176;C in a sealed bag with a desiccant until needed. 
	NOTE: Prior to using the tip, it should be brought to room temperature before opening the sealed bag and taking it out. The moisture drastically reduces the reactivity of lectins.</li>
</ol>
2. Preparation of the analytes
<ol>
	<li>Prepack the biotinylation reagent for routine analysis. 
	NOTE: For routine analysis, it is helpful to prepack the biotinylation reagent (Biotin-(AC5)2 Sulfo-Osu, see Table of Materials) in small portions as follows for quick analysis. It is recommended that the biotinylation reagent be dried and stored, as its succinimidyl ester is generally susceptible to hydrolysis.
	<ol>
		<li>Dissolve the biotinylation reagent in deionized water.</li>
		<li>Dispense 10 &#181;g of the reagent into tubes using a pipette.</li>
		<li>Dry the tubes with biotinylation reagent in a refrigerated vacuum concentrator and store them with a desiccant at 4 &#176;C in a lightproof bag until use.</li>
	</ol>
	</li>
	<li>Add the analyte solution (200 ng eq. protein) to the tube containing the dried biotinylation reagent prepared in step 2.1. 
	NOTE: Be mindful of the buffer composition used to dissolve and dilute the analytes, depending on the properties of the biotinylation reagent used. For example, avoid using primary amine reagents as buffers for amine coupling.This will prevent a reduction in the biotinylation efficiency of the analytes.</li>
	<li>Mix well by vortexing, then spin down (1000 x g for 5 s at room temperature) in a tabletop centrifuge to drop the liquid on the lid to the bottom of the tube.</li>
	<li>Incubate the tubes for 1 h at room temperature, shielded from light.</li>
	<li>Dilute the biotinylated samples 10-fold in the Probing buffer (1% Triton X-100, 0.5 M Glycine, 1 mM CaCl2, and 1 mM MnCl2 in TBS) to inactivate any unreacted biotinylation reagents. 
	NOTE: In the case of reactions by amine coupling, the unreacted biotinylation reagent is consumed by the reaction with a buffer containing a primary amine reagent such as Tris in TBS, which prevents the biotinylation of lectins and other substances immobilized on the beads in the subsequent measurement.</li>
	<li>Incubate at room temperature, shielded from light, for 2 h.</li>
	<li>Assess the biotinylation efficiency of the analytes through Western blotting, using HRP-labeled streptavidin as a detection probe if necessary.Please refer to the literature for Western blotting methods6.</li>
</ol>
3. Preparation of reagents for glycan profiling with the automated reaction measurement device
<ol>
	<li>Dilute the HRP-conjugated streptavidin with the blocking solution in a 1:3000 ratio.</li>
	<li>Mix with equal volumes of Substrate A and Substrate B (see Table of Materials). Use 160 &#181;L of mixed substrate per sample.</li>
	<li>Dispense the blocking solution, wash buffer (0.1% Triton X-100/TBS), TBS, HRP-conjugated streptavidin solution, and each substrate into the designated wells of a cartridge with 10 liquid reservoirs. 
	NOTE: The recommended volume and placement of solutions to fill each well of the reaction cartridge are described below. However, the solutions and well arrangement can be changed in the automated reaction measurement device as appropriate in advance.
	<ol>
		<li>Add 0.2 mL of HRP-labeled streptavidin prepared in step 3.1. to well #2 using a pipette. The well is named #1 from the front of the cartridge.</li>
		<li>Add 0.2 mL of blocking solution to well #3 using a pipette.</li>
		<li>Add 1 mL of TBS to wells #4 and #6 using a pipette.</li>
		<li>Add 1 mL of wash buffer to wells #7 and #8 using a pipette.</li>
		<li>Add 0.16 mL of substrate mixture to well #10 using a pipette.</li>
		<li>Confirm that wells #1, #5, and #9 are empty.</li>
	</ol>
	</li>
	<li>Add the analytes prepared in step 2 to a 2 mL-protein-low adsorbent screwcap microtube.</li>
	<li>Open the front door of the automated reaction measurement device.</li>
	<li>Set the liquid reservoirs prepared in step 3.3, the tips prepared in 1, and the analytes prepared in step 3.4 in the designated positions of the automated reaction measurement device (Figure 2D) as follows.Each measurement is performed in a single vertical line direction.
	<ol>
		<li>Place one tip prepared in step 1 in each hole at the position &#34;a&#34; yellow symbol in Figure 2D. 
		NOTE: Again, if refrigerated, bring the tips to room temperature before opening the sealed bag. Lectins lose their signal dramatically due to moisture.</li>
		<li>Remove the lid of the tube containing the analyte prepared in step 3.4 and insert it into each hole at position &#34;b&#34; similarly.</li>
		<li>Insert the cartridge containing the liquid prepared in step 3.3, being careful not to spill it at position &#34;c&#34;.</li>
		<li>Remove the entire &#34;waste box&#34; in Figure 2D when it is full, dump the contents into the trash bin, and return the &#34;waste box&#34; to its original position. 
		NOTE: The reacted tips are automatically collected in the &#34;waste box&#34; after measurement.</li>
	</ol>
	</li>
</ol>
4. Glycan profiling with the automated reaction measurement device
<ol>
	<li>Turn on the power switch on the side of the main unit.</li>
	<li>Touch the &#34;Start Diagnosis&#34; button displayed on the screen (Figure 3A). The self-diagnostic program starts automatically. After a while, the self-diagnostic program is completed, and the HOME screen appears (Figure 3B). 
	NOTE: The following operations can be performed by touching the respective icons. &#34;Assay&#34; to start measurement. &#34;Maintenance&#34; to confirm any system error. &#34;History&#34; to view past measurement data.</li>
	<li>Touch &#34;Assay&#34; displayed on the HOME screen to select the protocol (Figure 3B).</li>
	<li>Select the &#34;BAssaySTD&#34; of the assay method (Figure 3C) and then press &#34;Next&#34; on the lower right.</li>
	<li>Enter the sample name (Figure 3D) for each sample as necessary and then touch &#34;Next&#34; on the lower right.</li>
	<li>Enter the tip name (Figure 3E) used for each sample as necessary and then touch &#34;Next&#34; on the lower right.</li>
	<li>Confirm the notes to the right (Figure 3F), and if there is no problem, touch &#34;Start&#34; on the lower right for measurement.</li>
	<li>Confirm the screen switch to the running mode (Figure 3G) and wait until the &#34;finalize&#34; arrow is highlighted. If necessary, touch &#34;Break&#34; to stop running mode. 
	NOTE: The operations automatically controlled by the automated reaction measurement device are as follows: (1) Auto-micropipette nozzles inside the instrument attach a maximum of eight -tips simultaneously (see the symbol yellow &#34;a&#34; in Figure 2D); (2) Repeatedly aspirate/discharge the solution in the tube containing the analyte for 5 min; (3) Transfer to the blocking solution and aspirate/discharge for 5 min; (4) Transfer to TBS and aspirate/discharge for 2 min; (5) Transfer to the HRP-labeled streptavidin solution and aspirate/discharge 5 min; (6) Transfer to the wash buffer and aspirate/discharge for 7 min; (7) Transfer to TBS and aspirate/discharge for 2 min; (8) Transfer to substrate mixture and aspirate at once; (9) Scan the chemiluminescence of eight tips simultaneously at the back of the device to obtain peak data; (10) Drop used tips into the waste box; (11) Nozzle returns to the initial position.</li>
	<li>Confirm that the screen has changed to the result mode (Figure 3H) when the measurement is completed. The eight dotcodes are simultaneously displayed on the screen for easy verification. 
	NOTE: Touch the &#34;History&#34; on the lower left to display the Scan chart (Figure 3I,J), bar graph (Figure 3K,L), table (Figure 3M), and detail (Figure 3N) as the result on the screen. View the Results from other lanes from the drop-down &#34;Lane&#34; list.</li>
	<li>Insert a USB memory into the USB port on the side of the main unit and touch &#34;Export&#34; on the lower right of the result mode to save the data of all lanes individually in the USB memory.</li>
	<li>Export the raw data (Figure 3M) as a CSV file using a USB port for further data analysis and individual graphing in Excel. &#34;Value&#34; of the table denotes the signal intensity of each lectin. The values of intensity represent the maximum value of the peak data (Figure 3I,J) in the scan of reacted tips. 
	NOTE: The peak extraction threshold can be set. Depending on the analytes, the signal in the spacer bead portion may be higher overall, in which case the value in the spacer portion is subtracted as background.</li>
</ol>

Automated Lectin-Based Glycan Profiling Using Bead Array in a Single Tip (BIST)

<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>

AO, TO, NT, HS, KK, MA, SY, TM, KN, OS, and KS are employees of Precision System Science Co., Ltd.

In this study, a rapid evaluation technique for glycosylation employing "bead array in a single tip" technology has been developed. The present study introduced a standard GlycoBIST tip, designed for both glycoscience and non-glycoscience researchers, to facilitate routine, comprehensive glycosylation evaluation. The lectin microarray, typically employing 20-100 lectins12,13, has been extensively utilized in evaluations. However, considering the overlapping specificities of some lectins on the microarray and the relatively limited variety of glycoforms on a target glycoprotein compared to glycomics of crude clinical samples, it was anticipated that 15 lectins would suffice for simplified evaluation of focused glycan profiling.
The critical step in the protocol is to allow the lectin-immobilized beads or tips filled with those beads to return to room temperature after storage before using them for measurements. In particular, it was observed that condensation weakens the signals; therefore, it is advised to never open the storage bag until it returns to room temperature. For the storage of biotinylation reagents, it is also essential to completely dry before storage. An insufficient drying process decreases the efficiency of biotinylation to protein owing to the hydrolysis of the biotinylation reagent14.
Regarding modifications and troubleshooting, a method was explored to reduce variability, especially pertaining to the measurement method in the automated reaction measurement device. In particular, when the volume of buffer in the reservoir used for analysis is 150 &#181;L, bubbles enter the tip, leading to reduced reaction efficiency and resulting in a large variation in values. Therefore, it is recommended that at least 200 &#181;L of buffer be placed in the reservoir. 
The amount of liquid that fills the tips during auto-pipetting is also an important factor. The tips cannot completely expel the liquid as it moves to the next reaction step. Therefore, in no small amount, the buffer from the previous step is carried over into the next step, and the previous buffer remains at the top area of the inserted solution when the new buffer is aspirated. Therefore, the substrate and other solutions should be sufficiently aspirated to fill the top reservoir (see Figure 1A). Given the irreversibility of antibody reactions on the instrument, it is crucial to exercise caution and avoid errors during the initial setup.
One limitation of the method is that it is restricted to soluble proteins derived from serum and culture supernatants. In the current method, lectin is not covalently crosslinked to the bead, rendering it unable to analyze samples containing high concentrations of surfactants, such as cell and tissue extracts. Consequently, future improvements are necessary to address these limitations.
The significance of the lectin bead array lies in the interchangeability and expandability of the lectin species fixed on the beads. For example, in large-scale differential analysis (&gt;1,000 samples) using the automated glycan profiling system, users can tailor a 15-lectin lineup in the tip based on preliminary lectin microarray-based glycan profiling of the target glycoprotein (&lt;100 samples). Moreover, the high stability of the lectin-fixed beads enables immediate experimentation from the initial tip design to measurement with routine procedures. Not only the 25 of 28 lectins that have demonstrated high reliability (Table 1), but any lectins of interest to the user can be used to create a custom automated glycan profiling system for their specific experiments, following the aforementioned routine reliability test. This approach led to the design of a possible 120-lectin bead array of eight different types of tips for expanded measurement in conjunction with the standard glycan profiling system.
A previous study focused on establishing a detection procedure for Cy3-labeled glycoproteins in line with the lectin microarray15. This method was limited to 13 lectins in parallel in the tip because of fluorescence interference. The current method, using HRP detection labels, accommodates 15 lectin-fixed beads and two control beads (positive and negative) in a tip. In addition, the biotinylation of samples has shortened the analysis process.
This analytical method can be applied not only to academic research, but also to medical and pharmaceutical research, food, cosmetics, and other industrial fields. While lectin microarray and glycoproteomics technologies are well-established, the approach of this study remains significant for its full automation and capability for rapid analysis with fewer steps. In the future, enhancements to this analysis will be made to a quantitative method by normalizing the signal using certain glycoproteins, employing antibody-fixed beads alongside lectin-fixed beads in a tip.

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.

<table><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&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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, a critical post-translational modification, influences the stability, efficacy, and immunogenicity of recombinant proteins, including biopharmaceuticals. Glycan structures exhibit significant heterogeneity, varying with production cell types, culture conditions, and purification methods. Consequently, monitoring and evaluating the glycan structures of recombinant proteins is vital, particularly in biopharmaceutical production. The lectin microarray, a technique complementary to mass spectrometry, boasts high sensitivity and ease of use. However, it typically requires more than a day to yield results. To adapt it to non-glycoscience research or drug product process development, an automated, high-throughput alternative is needed. Therefore, the world's first fully automated lectin-based glycan profiling system was developed, utilizing the "bead array in a single tip (BIST)" technology concept. This system allows for the preparation and storage of lectin-immobilized beads in units of 1,000, with customizable parallel insertion orders for various purposes. This article presents a practical protocol for research involving "glyco-qualified" recombinant proteins. After testing their reactivity against 12 polyacrylamide-glycan conjugates, 15 lectins were selected to increase the system's versatility. In addition, the sample labeling process was optimized by switching from Cy3 to biotin, reducing the overall processing time by 30 min. For immediate data qualification, lectin-binding signals are displayed as a dotcode on the top monitor. The system's reliability was confirmed through day-to-day reproducibility tests, repeatability tests, and long-term storage tests, with a coefficient of variation of &lt;10%. This user-friendly and rapid glyco-analyzer has potential applications in the quality monitoring of endogenous glycoproteins for biomarker evaluation and validation. This method facilitates analysis for those new to glycoscience, thereby broadening its practical utility.

First, the signal distribution is shown for each lectin to understand the characteristics of the lectin bead array. A library of lectin-fixed beads (1,000 beads each) was prepared. Fifteen beads were randomly picked from the 1,000 and added to a single tip. A typical chart is shown at the bottom of Figure 1A, representing almost the same signal intensity observed among the 15 beads immobilizing the same lectin. This measurement procedure can be applied to qualify hand-made lectin bead production prior to the use of glycan profiling. For instance, six tips are used for the simultaneous measurement to calculate the coefficient of variation (CV) from the signals on the 90 beads. A typical example is shown in Table 1. When the lectin-fixed beads library was expanded to accommodate 28 different lectins, 25 out of 28 lectins demonstrated high reproducibility with a CV of less than 10%.
Next, the data for a lectin bead array with 15 different lectins are shown. The 15 lectins shown in Table 2 were selected for the lectin bead array. Additionally, 12 polyacrylamide (PAA) mono/di/trisaccharides listed in Table 3 were measured. After assessing the measurement reliability using a dotcoding display on the screen, the raw data were exported from the automated reaction measurement device (see protocol step 4.11), and a graph was created in Excel using the reaction peak values at each saccharide (Figure 4A). These values were then used for principal component analysis to visualize the correlation between the selected 15 lectins and the reacted saccharides (Figure 4B,C). This resulted in a distinct difference in the binding pattern of each lectin against the 12 PAA-sugars shown in Figure 4A. Herein, the validity of each signal is shown to refer to integrated previous lists of lectin and saccharide specificities7,8 (also refer to these specificities in Table 2). As AAL binds to terminal &#945;-Fuc, Sia-Lex, and Lex, automatic glycan analysis confirmed AAL&#39;s recognition of fucose-containing molecules (Figure 4A). SSA and MAL (see Table 2 for the full names of lectins and given abbreviations) recognized Sia&#945;2-6Gal/GalNAc and Sia&#945;2-3Gal, respectively. MAL in the bead array can also bind to 3&#39;-O-sulfo-Gal&#946;3GalNAc and LacNAc. This is consistent with previous results analyzed using machine learning7, in which MAL prefers the 3&#39;-O-sulfo-Gal dominantly, whereas LacNAc is not dominant9. RCA120, a Gal-recognizing lectin, strongly reacted with terminal &#946;-Gal, including Lac/LacNAc, and was permitted to bind to Sia&#945;2-6Lac but not Sia&#945;2-3Lac. ECA had reactivity with LacNAc much higher than Lac. WFA had a broader specificity, which recognized not only &#945;/&#946;-GalNAc but also terminal &#946;-Gal as reported7,10. GSL II, which recognizes GlcNAc and a galactosylated tri/tetra-antennary N-glycans, did not bind to any saccharides except for a single GlcNAc among the prepared analytes. GRFT, which recognizes high mannose-type N-glycans, reacted with &#945;-Man. ABA, Jacalin, and MAH have been generally used to detect O-glycans. As ABA prefers the Gal&#946;1-3GalNAc&#945;-Thr/Ser (T) and sialyl-T structures, ABA in the bead array strongly reacted with 3&#39;-O-sulfo-Gal&#946;1-3GalNAc and weakly bound to &#945;-GalNAc. ABA also recognized &#946;-GlcNAc, consistent with the previous reports on binding to agalactosylated N-glycans7. Jacalin has a relatively broader specificity and thus reacted with &#945;-mannose, &#946;-GalNAc, &#946;Gal, &#945;-GalNAc, 3&#39;-O-sulfo-Gal&#946;1-3GalNAc. MAH specifically recognized 3&#39;-O-sulfate-Gal&#946;1,3GalNAc as previously reported7. The residual four lectins, NPA, LCA, PHA-L, and PHA-E, recognized the internal structure of N-glycan not included in the prepared PAA saccharides and thus had no affinity with all analytes.
Using the obtained data, the similarity of lectins contributing to the binding to each saccharide can be clarified by plotting them on a principal component analysis graph (Figure 4B). All of the saccharides that contributed to the variance in Figure 4B were represented by eigenvector, a method of statistical analysis in which the contribution ratio is represented by a vector (Figure 4C). It is noteworthy here that the reacted saccharides contribute to each lectin without bias. Lectins do not exhibit specificity for only one type of saccharide. For example, the presence of complex-type glycans is determined by the partial recognition of some glycans within complex-type glycans in humans. Jacalin, which recognizes O-glycans (see the specificities in Table 2), can recognize the 3&#39;suTF structure and &#945;-GalNAc among the saccharides used in this study, and therefore, the 3&#39;suTF and &#945;-GalNAc were plotted (Figure 4C) against the position direction of Jacalin (brown dot) plotted in Figure 4B. WFA can recognize &#945;/&#946;-GalNAc among the saccharides used in this study; therefore, &#945;/&#946;-GalNAc in Figure 4C was plotted in the same direction as the WFA plotted in Figure 4B. In contrast, &#945;-GalNAc is plotted between WFA and Jacalin because it is a saccharide structure that Jacalin is capable of binding to easily. All 11 saccharides plotted in this study are consistent with the specificity (saccharide binding) of lectins reported previously7,8, indicating that the proposed method is a reliable measurement method. Furthermore, the eigenvectors corresponding to each saccharide are dispersed, supporting that the 15 lectins were selected with minimal bias to ensure comprehensive coverage in the analysis. Consequently, the tip replenished with these 15 lectins was defined as the standard GlycoBIST-tip (Table 2).
In the more practical example, purified protein products were subjected to the tip measurement (Figure 5). Bovine thyroglobulin, which has an N-glycosylation site and contains complex, hybrid, and high mannose-type N-glycans11, was analyzed. The standard tip indicated increased reactivity of certain lectins to the saccharides8 (please refer to the specificities in Table 2): PHA-E, which recognizes bisecting GlcNAc; GRFT, recognizing mannose-type N-glycans; SSA, recognizing Sia&#945;2-6Gal/GalNAc; RCA120, recognizing lactose and LacNAc; and AAL, recognizing fucose. These glycan structures are present in complex, hybrid, and high mannose-type N-glycans, suggesting that the N-glycan structure of thyroglobulin11 could be effectively evaluated. Furthermore, thyroglobulin treated with sialidase A, an enzyme that digests sialic acid, showed decreased reactivity with SSA, which recognizes sialic acid, and increased reactivity with RCA120, WFA, and ECA, which are more readily recognized after the removal of sialic acids. The presence of O-glycan structures in bovine thyroglobulin has not been previously reported. As expected, there was no recognition of O-glycan in thyroglobulin with or without sialidase treatment in the automatic glycan analysis.
A within-run reproducibility test using the 15 selected lectins is shown to understand the robustness of the measurement (Table 4). For this test, seven standard tips for analytes and an additional tip for negative control were prepared, and all eight tips were measured simultaneously. This procedure was repeated three times in a single day. &#34;Mix Analytes,&#34; a combination of analytes that led to obtaining significant signals on all the lectins, were prepared for the quality control of the beads array. The Mix Analytes were formulated by appropriately mixing sialidase-digested erythropoietin (EPO), sialidase- and galactosidase-digested EPO, human IgA, matrix metalloproteinase 3 (MMP3), and thyroglobulin. Each glycoprotein (200 ng) was labeled with 10 &#181;g of biotinylation reagent in PBS containing Triton X-100. If necessary, sialidase and galactosidase treatments were performed as described in the instructions. After digestion, the products were incubated in a heat block at 75 &#176;C for 10 min to inactivate the enzyme. The appropriate amounts of biotinylated glycoproteins (3 ng of EPO, 8 ng of human IgA, 5 ng of MMP3, and 15 ng of thyroglobulin) were mixed immediately before analysis. The measurement results indicated that the maximum CV value was 13.5%, and the average CV for the 15 lectins was 8.2%, demonstrating high reproducibility.
Additionally, a day-to-day reproducibility test is shown to understand variations due to the measurement date (Table 5). Standard tips and Mix Analytes were prepared in advance, and measurements of seven tips were repeated daily for five consecutive days. The results showed that most lectins had a CV of less than 10%. However, some lectins, such as LCA and ECA, exhibited higher CVs. The average CV of the 15 lectins per cycle was up to 7.7%, and the average CV values over 5 days were less than 10%, signifying high reproducibility. It was noted that Jacalin, owing to the self-digestion of MMP3 in the Mix Analyte, demonstrated low temporal reproducibility. Therefore, a more suitable analyte should be identified to replace MMP3.
To understand the stability of lectin-fixed beads, a long-term stability test is shown using these dried lectin-fixed beads (Table 6). Standard tips, assay reagents, and Mix Analytes were prepared in advance, and measurements were performed after 12 months of storage. The aforementioned five types of biotinylated analytes were stored individually and mixed prior to measurement. The results showed that the average CV for the 15 lectins was less than 10%, even up to 12 months after dry storage. This suggests the feasibility of rapid and precise measurements by storing numerous tips (up to 1,000) from the same lot at once.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66571/66571fig01v2.jpg" /> 
Figure 1: Schemes of the &#34;bead array in a single tip&#34; instrument and analysis method. (A) Scheme of automatic glycan profiling system. Lectin-fixed beads can be dried and stored in tips. When tips and cartridges containing reagents like HRP-labeled anti-streptavidin antibody (SA-HRP) are set in the measuring instrument (automated reaction measurement device) and activated, the tip functions as an autopipette. The chemiluminescence detection scanner at the instrument&#39;s rear quantifies signals from eight tips simultaneously. Quantitative data are displayed as dot codes on the instrument&#39;s touchscreen for rapid confirmation of measurement results. The lower section shows the results of measuring 90 beads with the same lectin. The measurement results are transported from the instrument and graphed by the individual researcher. (B) Scheme of the detection method used in this experiment. Target proteins are pre-biotinylated and detected using SA-HRP. <a href="https://www.jove.com/files/ftp_upload/66571/66571fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66571/66571fig02v2.jpg" /> 
Figure 2: Images of materials and setup for automatic glycan profiling with the automated reaction measurement device. (A) Depiction of lectin-fixing beads being packed into a tip. Picking up the beads with antistatic tweezers on the antistatic mat and filling the tip with the beads. (B) Pinching the tip with a nipper. A slight upper portion of the top bead is squeezed. (C) A nipper for crimping the tips. The plastic (yellow circle) is pinched to prevent them from snipping completely. (D) Arrangement of materials used in the automated reaction measurement device. a: tips, b: tube containing analytes, and c: cartridges containing liquid should be inserted in place, respectively. In the waste box, the reacted tips are collected after measurement. The box can be removed. <a href="https://www.jove.com/files/ftp_upload/66571/66571fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66571/66571fig03.jpg" /> 
Figure 3: Touch screen for machine manipulation. (A) The self-diagnostic program starts automatically when the power is turned on. (B) The HOME screen displays &#34;Assay,&#34; &#34;Maintenance,&#34; and &#34;History&#34;. (C-G) Operation of the &#34;Assay&#34; mode. Assay method selection (C). Multiple protocols can be registered in advance. Input of sample (analyte) order (D). Selecting the type of the tip (E). Confirmation of the assay method settings (F) and starting the assay from the &#34;Start Assay&#34; screen (G). (H-N) Representation of the result in &#34;History.&#34; Lectin dotcode (H) displayed after assay completion or from &#34;History&#34; mode. A scan chart of 15 lectins is displayed on the screen for all samples (I) or for each sample (J). The scan chart is displayed as a bar graph (K). Bar charts for other lanes can be selected from the drop-down &#34;Lane&#34; list. Bar graph comparing eight lanes of samples per lectin (L). Other lectins can be selected from the dropdown &#34;Lectin&#34; list. Table showing quantitative values (M). Record of the executed measurement method (N). <a href="https://www.jove.com/files/ftp_upload/66571/66571fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66571/66571fig04.jpg" /> 
Figure 4: Evaluation of reactivity with PAA saccharides in the automatic glycan profiling system. (A) A graph depicting the reactivity of lectins with various PAA saccharides was adjusted for negative control values. (B) Principal component analysis based on the data in (A). (C) Eigenvectors from the analysis in (B). Compatible analysis software was used for parts (B) and (C). <a href="https://www.jove.com/files/ftp_upload/66571/66571fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66571/66571fig05.jpg" /> 
Figure 5: Verification of GlycoBIST reactivity using purified bovine thyroglobulin protein. Sample preparation without treatment was conducted under the same digestion conditions as the buffer alone, excluding the addition of sialidase A. <a href="https://www.jove.com/files/ftp_upload/66571/66571fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Repeatability test with lectin-fixed beads. The repeatability of reactivity for 28 lectins was evaluated. Analytes were reacted with each tip. EPO: erythropoietin, PSA: prostate-specific antigen, Tf: transferrin, M2BP: Mac-2 binding protein, hIgG: human IgG, MMP3: matrix metalloproteinase 3, (Sia+): sialidase A-digested analytes, (Sia+, Gal+): sialidase A and galactosidase-digested analytes, Ab: antibody. DSA: Datura stramonium agglutinin, HypninA2: Hypnea japonica agglutinin, WGA: wheat germ agglutinin, UDA: Urtica dioica agglutinin, BPL: Bauhinia purpurea lectin, Orysata: Oryza sativa lectin, LSL-N: Laetiporus sulphureus lectin N-terminal domain, SNA: Sambucus nigra lectin, BanLec: banana lectin, MPA: Maclura pomifera agglutinin, TxLC-I: Tulipa gesneriana agglutinin, AOL: Aspergillus oryzae lectin, ACG: Agrocybe cylindracea galectin. CV: coefficient of variation. <a href="https://www.jove.com/files/ftp_upload/66571/66571_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: List of lectins constituting the standard tipfor automatic glycan profiling. <a href="https://www.jove.com/files/ftp_upload/66571/66571_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: PAA saccharides used as standards for automatic glycan profiling system. <a href="https://www.jove.com/files/ftp_upload/66571/66571_Table 3.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4: Results of the within-run reproducibility test. CV: coefficient of variation. <a href="https://www.jove.com/files/ftp_upload/66571/66571_Table 4.xlsx" target="_blank">Please click here to download this Table.</a>
Table 5: Results of the between-day reproducibility test. CV: coefficient of variation. <a href="https://www.jove.com/files/ftp_upload/66571/66571_Table 5.xlsx" target="_blank">Please click here to download this Table.</a>
Table 6: Results of the long-term stability test. CV: coefficient of variation. <a href="https://www.jove.com/files/ftp_upload/66571/66571_Table 6.xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Advancing Protein Glycosylation Research Using a Fully Automated System

A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

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, ...

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

Research

JoVE Journal

Biochemistry

707 Views.  National Institute of Advanced Industrial Science and Technology. A system for the automated and rapid analysis of glycans on proteins was previously developed. This article presents the protocol for "glyco-qualitative analysis," optimized for a broad spectrum of users, such as those engaged in analyzing glycan structures in biopharmaceuticals and other glycoconjugate materials.

Video: Author Spotlight: Advancing Protein Glycosylation Research Using a Fully Automated System

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Chemistry

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Bioengineering

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

Immunology and Infection

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Biology

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease ...

Medicine

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...