This work was supported by grants from National Natural Science Foundation of China (81673247 &#38; 81872682) and Australian-China Collaborative Grant (NH &#38; MRC - APP1112767 -NSFC 81561128020).

All the subjects included in the protocol have been approved by the Ethics Committee of the Capital Medical University, Beijing, China12. Written informed consent was obtained from each subject at the beginning of the study.
1. IgG isolation
<ol>
	<li>Prepare the chemicals including binding buffer (phosphate buffered saline, PBS): 1x PBS (pH = 7.4), neutralizing buffer: 10x PBS (pH = 6.6-6.8), eluent: 0.1 M formic acid (pH = 2.5), neutralizing solution for eluent: 1 M ammonium bicarbonate, stored buffer: 20% ethanol + 20 mM Tris + 0.1 M NaCl (pH = 7.4), cleaning solution for protein G: 0.1 M NaOH + 30% propan-2-ol. 
	NOTE: The level of pH is critical in this protocol. The elution of IgG requires a very low pH, and there is a risk of the loss of sialic acids due to acid hydrolysis. Therefore, elution occurs within seconds, and the pH is quickly restored to neutrality, preserving the integrity of IgG and the sialic acids.</li>
	<li>Prepare the samples: thaw the frozen plasma sample then centrifuge at 80 x g for 10 min, and leave the protein G monolithic plate and the abovementioned chemicals for 30 min at room temperature (RT).</li>
	<li>Transfer a 100 &#181;L sample (which can be used to detect 2x to prevent the first failure) into a 2 mL collection plate (here, a total of six standard samples, one control sample [ultra-pure water], and 89 plasma samples were designed for 96 well plates and randomly assigned to the plate).</li>
	<li>Dilute the samples with 1x PBS by 1:7 (v/v).</li>
	<li>Clean a 0.45 &#181;m hydrophilic polypropylene (GHP) filter plate with 200 &#181;L of ultra-pure water (repeat 2x).</li>
	<li>Transfer the diluted samples into the filter plate and filter the samples into the collection plate using a vacuum pump (control vacuum pressure at 266.6-399.9 Pa).</li>
	<li>Preparation of the protein G monolithic plates
	<ol>
		<li>Discard the storage buffer.</li>
		<li>Clean the monolithic plates with 2 mL of ultra-pure water, 2 mL of 1x PBS, 1 mL of 0.1 M formic acid, 2 mL of 10x PBS, 2 mL of 1x PBS (sequentially), and remove flowing liquid using a vacuum pump.</li>
	</ol>
	</li>
	<li>Transfer the filtered samples to the protein G monolithic plate for IgG binding and cleaning, then clean the monolithic plates with 2 mL of 1x PBS (repeat the cleaning 2x).</li>
	<li>Elute IgG with 1 mL of 0.1 M formic acid and filter the samples into the collection plate by vacuum pump, then add 170 &#181;L of 1 M ammonium bicarbonate into the collection plate.</li>
	<li>Detect IgG concentration using an absorption spectrophotometer (optimal wavelength = 280 nm).
	<ol>
		<li>Open the software and select the protein-CY3 mode.</li>
		<li>Draw 2 &#181;L of ultra-pure water and load it into the screen, then click Blank in the software to clear the screen (repeat 1x).</li>
		<li>Draw 2 &#181;L of ultra-pure water and load it into the screen, then click Sample in the software to detect the ultra-pure water.</li>
		<li>Draw 2 &#181;L of IgG sample and load it into the screen, then click Sample in the software to detect the sample.</li>
		<li>Draw 2 &#181;L of ultra-pure water and load it into the screen, then click Blank in the software to clear the screen.</li>
		<li>Close the software. 
		NOTE: The formula for calculating IgG concentration is as follows: 
		 
		CIgG = absorbance x extinction coefficient (13.7) x 1,000 &#181;g/mL</li>
	</ol>
	</li>
	<li>Put the extracted IgG to dry in an oven at 60 &#176;C and preserve the extracted IgG (300 &#181;L extracted IgG for 4 h).
	<ol>
		<li>Remove 300 &#181;L of extracted IgG if the concentration is greater than 1,000 &#181;g/mL.</li>
		<li>Remove 350 &#181;L of extracted IgG if the concentration is between 500-1,000 &#181;g/mL.</li>
		<li>Remove 400 &#181;L of extracted IgG if the concentration is between 200-500 &#181;g/mL.</li>
		<li>Remove 600 &#181;L of extracted IgG if the concentration is smaller than 200 &#181;g/mL. 
		NOTE: The concentration of IgG should be preferably &#62;200 &#181;g/mL for subsequent detection. The average amount of IgG should be preferably &#62;1,200 &#181;g, which can be tested 2x in case the first test fails.</li>
	</ol>
	</li>
	<li>Cleaning the protein G monolithic plate for reuse
	<ol>
		<li>Wash the plate with 2 mL of ultra-pure water, 1 mL of 0.1 M NaOH (for removing precipitated proteins), 4 mL of ultra-pure water, and 4 mL of 1x PBS (sequentially), then remove flowing liquid using a vacuum pump.</li>
		<li>Wash the plate with 2 mL of ultra-pure water, 2 mL of 30% propan-2-ol (for removing bound hydrophobic proteins), 2 mL of ultra-pure water, and 4 mL of 1x PBS (sequentially), then remove flowing liquid using a vacuum pump.</li>
		<li>Wash the plate with 1 mL of buffer (20% ethanol + 20 Mm Tris + 0.1 M NaCl) and add 1 mL of buffer (20% ethanol + 20 Mm Tris + 0.1 M NaCl) to the plate, then leave the plate at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Glycan release
<ol>
	<li>Prepare the dried IgG and store the chemicals including 1.33% SDS, 4% Igepal (store away from light), and 5x PBS at RT.</li>
	<li>Prepare PNGase F enzyme by diluting 250 U enzyme with 250 &#181;L of ultra-pure water.</li>
	<li>Denaturation of IgG
	<ol>
		<li>Add 30 &#181;L of 1.33% SDS and mix by vortexing, transfer the sample into a 65 &#176;C oven for 10 min, then remove it from the oven and let rest for 15 min.</li>
		<li>Add 10 &#181;L of 4% Igepal and place it on the shaking incubator for 5 min.</li>
	</ol>
	</li>
	<li>Removal and release of glycans
	<ol>
		<li>Add 20 &#181;L of 5x PBS and 30-35 &#181;L of 0.1 mol/L NaOH to regulate a pH of 8.0, and mix by vortexing. Add 4 &#181;L of PNGase F enzyme and mix by vortexing. Then, incubate for 18-20 h in a 37 &#176;C water bath.</li>
		<li>Put the released glycans to dry in an oven at 60 &#176;C for 2.5-3.0 h.</li>
		<li>Save the released glycans at -80 &#176;C until further measurement. 
		NOTE: This step is critical. The key to glycan release is improving the activity of the PNGase F enzyme to maximize its efficiency.</li>
	</ol>
	</li>
</ol>
3. Glycan labeling and purification
<ol>
	<li>Prepare the 2-aminobenzamide (2-AB) labeling reagent with 0.70 mg of 2-AB, 10.50 &#181;L of acetic acid, 6 mg of sodium cyanoborohydride (NaBH3CN), and 24.50 &#181;L of dimethyl sulfoxide (DMSO) (total volume = 35 &#181;L). Then, add acetic acid, 2-AB, and NaBH3CN into the DMSO in order.</li>
	<li>Label the glycans using 35 &#181;L of 2-AB labeling reagent, transfer the labeled glycans to the oscillator for 5 min, transfer to the oven for 3 h at 65 &#176;C, then transfer to RT for 30 min. 
	NOTE: The entire glycan labeling step must be performed while protected from light.</li>
	<li>Pretreat a 0.2 &#181;m GHP filter plate with 200 &#181;L of 70% ethanol, 200 &#181;L of ultra-pure water, and 200 &#181;L of 96% acetonitrile (4 &#176;C), then remove waste using a vacuum pump.</li>
	<li>Purification of 2-AB labeled glycan
	<ol>
		<li>Add 700 &#181;L of 100% acetonitrile to the 2-AB labeled glycan and transfer to a shaking incubator for 5 min.</li>
		<li>Centrifuge at 134 x g for 5 min (4 &#176;C).</li>
		<li>Transfer the sample to a 0.2 &#181;m GHP filter plate for 2 min and remove the filtrate (flowing liquid) using a vacuum pump.</li>
	</ol>
	</li>
	<li>Wash 2-AB labeled glycan with 200 &#181;L of 96% acetonitrile (4 &#176;C) and remove the filtrate (flowing liquid) using a vacuum pump 5x-6x.</li>
	<li>Elute 2-AB labeled glycan with 100 &#181;L of ultra-pure water 3x.</li>
	<li>Transfer the 2-AB labeled glycan into an oven to dry at 60 &#176;C for 3.5 h.</li>
	<li>Save the labeled N-glycans at -80 &#176;C until further measurement.</li>
</ol>
4. Hydrophilic interaction chromatography and analysis of glycans
<ol>
	<li>Conditioning of UPLC instruments and preparation of mobile phases
	<ol>
		<li>Prepare mobile phases including solvent A: 100 mM ammonium formate (pH = 4.4), solvent B: 100% acetonitrile, solvent C: 90% ultra-pure water (10% methanol), and solvent D: 50% methanol (ultra-pure water).</li>
		<li>Open the software to control the mobile phases.</li>
		<li>Wash UPLC instruments at flow rate of 0.2 mL/min (50% solvent B and 50% solvent C) balancing for 30 min, then at a flow rate of 0.2 mL/min (25% solvent A and 75% solvent B) balancing for 20 min, then a flow rate of 0.4 mL/min balancing.</li>
	</ol>
	</li>
	<li>Dissolve the labeled N-glycans with 25 &#181;L of a mixture of 100% acetonitrile and ultra-pure water at a 2:1 ratio (v/v). Then, centrifuge at 134 &#215; g for 5 min (4 &#176;C) and load 10 &#181;L of the labeled N-glycans into the UPLC instruments.</li>
	<li>Separate the labeled N-glycans at flow rate of 0.4 mL/min with a linear gradient of 75% to 62% acetonitrile for 25 min. Then, perform an analytical run by dextran calibration ladder/glycopeptide column on a UPLC at 60 &#176;C (here, samples were kept at 4 &#176;C prior to injection).</li>
	<li>Detect N-glycan fluorescence at excitation and emission wave lengths of 330 nm and 420 nm, respectively.</li>
	<li>Integrate the glycans based on peak position and retention time.</li>
	<li>Calculate the relative value of each Glycan Peak (GP)/ all Glycan Peaks (GPs) (percentage, %) as follows: GP1: GP1/GPs*100, GP2: GP2/GPs*100, GP3: GP3/GPs*100, etc.</li>
</ol>

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	<li>Kolarich, D., Lepenies, B., Seeberger, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycomics,+glycoproteomics+and+the+immune+system.">Glycomics, glycoproteomics and the immune system.</a> Current Opinion in Chemical Biology. 16, 214 (2012).</li><li>Cummings, R., Pierce, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Challenge+and+Promise+of+Glycomics.">The Challenge and Promise of Glycomics.</a> Chemistry Biology. 21 (1), (2014).</li><li>Shade, K. T. C., Anthony, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+Glycosylation+and+Inflammation.">Antibody Glycosylation and Inflammation.</a> Antibodies. 2, 392 (2013).</li><li>Gudelj, I., Lauc, G., Pezer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+G+glycosylation+in+aging+and+diseases.">Immunoglobulin G glycosylation in aging and diseases.</a> Cellular Immunology. 333, 65 (2018).</li><li>Huffman, J. 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=Comparative+performance+of+four+methods+for+high-throughput+glycosylation+analysis+of+immunoglobulin+G+in+genetic+and+epidemiological+research.">Comparative performance of four methods for high-throughput glycosylation analysis of immunoglobulin G in genetic and epidemiological research.</a> Molecular Cellular Proteomics. 13, 1598 (2014).</li><li>Stockmann, H., Adamczyk, B., Hayes, J., Rudd, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+high-throughput+IgG-antibody+glycoprofiling+platform.">Automated, high-throughput IgG-antibody glycoprofiling platform.</a> Analytical Chemistry. 85, 8841 (2013).</li><li>Kristic, J., 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=Glycans+are+a+novel+biomarker+of+chronological+and+biological+ages.">Glycans are a novel biomarker of chronological and biological ages.</a> Journals of Gerontology. Series A, Biological Sciences Medical Sciences. 69, 779 (2014).</li><li>Nikolac, P. M., 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=The+association+between+galactosylation+of+immunoglobulin+G+and+body+mass+index.">The association between galactosylation of immunoglobulin G and body mass index.</a> Progress in Neuropsychopharmacology Biological Psychiatry. 48, 20 (2014).</li><li>Liu, 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=The+changes+of+immunoglobulin+G+N-glycosylation+in+blood+lipids+and+dyslipidaemia.">The changes of immunoglobulin G N-glycosylation in blood lipids and dyslipidaemia.</a> Journal of Translational Medicine. 16, 235 (2018).</li><li>Lemmers, R., 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=IgG+glycan+patterns+are+associated+with+type+2+diabetes+in+independent+European+populations.">IgG glycan patterns are associated with type 2 diabetes in independent European populations.</a> Biochimica Biophysica Acta General Subjects. 1861, 2240 (2017).</li><li>Wang, Y., 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=The+Association+Between+Glycosylation+of+Immunoglobulin+G+and+Hypertension:+A+Multiple+Ethnic+Cross-Sectional+Study.">The Association Between Glycosylation of Immunoglobulin G and Hypertension: A Multiple Ethnic Cross-Sectional Study.</a> Medicine (Baltimore). 95, e3379 (2016).</li><li>Liu, 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=Ischemic+stroke+is+associated+with+the+pro-inflammatory+potential+of+N-glycosylated+immunoglobulin+G.">Ischemic stroke is associated with the pro-inflammatory potential of N-glycosylated immunoglobulin G.</a> Journal of Neuroinflammation. 15, 123 (2018).</li><li>Russell, A. 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The authors have nothing to disclose.

UPLC serves as a relative quantitative analysis method5,15. The results indicate that UPLC is a stable detection technology with good reproducibility and relative quantitative accuracy. The amount of glycans in each peak is expressed as a percentage of the total integrated area using UPLC, which is the relative value. The relative quantification improves the comparability of test samples. In addition, 96 well protein G plates are used to purify IgG with 96 sample...

N-glycosylation of human proteins is a common and essential post-translational modification1 and may help predict the occurrence and development of diseases relatively accurately. Due to the complexity of its structure, it is expected that there are more than 5,000 glycan structures, providing great potential as diagnostic and predictive biomarkers for diseases2. N-glycans attached to immunoglobulin G (IgG) have been shown to be essential for IgG&#39;s function, and IgG N-glycosylation participates in the balance between the pro- and anti-inflammatory systems3. Differential IgG N-glycosylation is involved in disease development and progression, representing both a predisposition and functional mechanism involved in disease pathology. The inflammatory role of IgG N-glycosylation has been associated with aging, inflammatory diseases, autoimmune diseases, and cancer4.
With the development of detection technology, the following methods are most widely used in high throughput glycomics: hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography with fluorescence detection (UPLC-FLR), multiplex capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and liquid chromatography electrospray mass spectrometry (LC-ESI-MS). These methods have overcome previous shortcomings of low flux, unstable results, and poor sensitivity specificity5,6.
UPLC is widely used to explore the association between IgG N-glycosylation and certain diseases (i.e., ageing7, obesity8, dyslipidemia9, type II diabetes10, hypertension11, ischemic stroke12, and Parkinson&#39;s disease13). Compared to the other three abovementioned methods, UPLC has the following advantages5,14. First, it provides a relative quantitative analysis method, and the data analysis that involves total area normalization improves the comparability of each sample. Second, the cost of equipment and required expertise are relatively low, which makes it easier to implement and transform glycosylation biomarkers into clinical applications. Presented here is an introduction to UPLC so it can be more widely used.

<table><tbody><tr><td>2-aminobenzamide, 2-AB</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>96-well collection plate</td><td>AXYGEN</td><td></td><td></td></tr><tr><td>96-well filter plate</td><td>Pol</td><td>0.45 um GHP</td><td></td></tr><tr><td>96-well monolithic plate</td><td>BIA Separations</td><td></td><td></td></tr><tr><td>96-well plate rotor</td><td>Eppendorf Co., Ltd, Germany</td><td>T_1087461900</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Huihai Keyi Technology Co., Ltd, China</td><td></td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Shenggong Biological Engineering Co., Ltd, China</td><td></td><td></td></tr><tr><td>Ammonium formate</td><td>Beijing Minruida Technology Co., Ltd.</td><td></td><td></td></tr><tr><td>Constant shaking incubator/rocker</td><td>Zhicheng analytical instrument manufacturing co., Ltd, China</td><td>ZWY-10313</td><td></td></tr><tr><td>Dextran Calibration Ladder/Glycopeptide column</td><td>Watts technology Co., Ltd, China</td><td>BEH column</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>Disodium phosphate</td><td>Shenggong Biological Engineering Co., Ltd, China</td><td></td><td></td></tr><tr><td>Electric ovens</td><td>Tester instruments Co., Ltd</td><td>202-2AB</td><td></td></tr><tr><td>Empower 3.0</td><td>Waters technology Co., Ltd, America</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Huihai Keyi Technology Co., Ltd, China</td><td></td><td></td></tr><tr><td>Formic acid</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>GlycoProfile 2-AB Labeling kit</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>HCl</td><td>Junrui Biotechnology Co., Ltd, China</td><td></td><td></td></tr><tr><td>High-speed centrifuge</td><td>Eppendorf Co., Ltd, Germany</td><td>5430</td><td></td></tr><tr><td>Igepal</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>Low temperature centrifuge</td><td>Eppendorf Co., Ltd, Germany</td><td></td><td></td></tr><tr><td>Low temperature refrigerator</td><td>Qingdao Haier Co., Ltd</td><td></td><td></td></tr><tr><td>Manifold 96-well plate</td><td>Watts technology Co., Ltd, China</td><td>186001831</td><td></td></tr><tr><td>Methanol</td><td>Huihai Keyi Technology Co., Ltd, China</td><td></td><td></td></tr><tr><td>Milli-Q pure water meter</td><td>Millipore Co., Ltd, America</td><td>Advantage A10</td><td></td></tr><tr><td>NaOH</td><td>Shenggong Biological Engineering Co., Ltd, China</td><td></td><td></td></tr><tr><td>PH tester</td><td>Sartorius Co., Ltd, Germany</td><td>PB-10</td><td></td></tr><tr><td>Phosphate buffered saline, PBS</td><td>Shenggong Biological Engineering Co., Ltd, China</td><td></td><td></td></tr><tr><td>Pipette</td><td>Eppendorf Co., Ltd, Germany</td><td>4672100, 0.5-10&amp;mu;l &amp;amp; 10-100&amp;mu;l &amp;amp; 20-200&amp;mu;l &amp;amp; 1000&amp;mu;l</td><td></td></tr><tr><td>PNGase F enzyme</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Shenggong Biological Engineering Co., Ltd, China</td><td></td><td></td></tr><tr><td>Propan-2-ol</td><td>Huihai Keyi Technology Co., Ltd, China</td><td></td><td></td></tr><tr><td>SDS</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>Sodium chloride</td><td>Shenggong Biological Engineering Co., Ltd, China</td><td></td><td></td></tr><tr><td>Sodium cyanoborohydride (NaBH3CN)</td><td>Sigma, China</td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Shanghai Yuanxi instrument Co., Ltd</td><td>B-500</td><td></td></tr><tr><td>Transfer liquid gun</td><td>Smer Fell Science and Technology Co., Ltd, China</td><td>4672100</td><td></td></tr><tr><td>Tris</td><td>Amresco, America</td><td></td><td></td></tr><tr><td>Ultra-low temperature refrigerator</td><td>Thermo Co., Ltd, America</td><td>MLT-1386-3-V; MDF-382E</td><td></td></tr><tr><td>Ultra-performance liquid chromatography</td><td>Watts technology Co., Ltd, China</td><td>Acquity MLtraPerformance LC</td><td></td></tr><tr><td>Vacuum Pump</td><td>Watts technology Co., Ltd, China</td><td>725000604</td><td></td></tr><tr><td>Volatilizing machine/Dryer</td><td>Eppendorf Co., Ltd, Germany</td><td>T_1087461900</td><td></td></tr><tr><td>Vortex</td><td>Changzhou Enpei instrument Co., Ltd, China</td><td>NP-30S</td><td></td></tr><tr><td>Water-bath</td><td>Tester instruments Co., Ltd</td><td>DK-98-IIA</td><td></td></tr><tr><td>Weighing balance</td><td>Shanghai Jingke Scientific Instrument Co., Ltd.</td><td>MP200B</td><td></td></tr></tbody></table>

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.

As shown in Figure 1, IgG N-glycans were analyzed into 24 initial IgG glycan peaks (GPs) based on peak position and retention time. The N-glycan structures are available through mass spectrometry detection according to a previous study (Table 1)15. To ensure that the results were comparable, total area normalization was applied, in which the amount of glycans in each peak was expressed as a percentage...

Watch this Scientific Journal Video about Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography at JoVE.com

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

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 ...

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Medicine

8.4K Views.  Capital Medical University. Ultra-performance vloeibare chromatografie is gevestigde technologie voor nauwkeurige analyse van IgG n-glycan Vanwege de gevoeligheid lijkt te zijn en het heeft de mogelijkheid om een specifieke informatie van glycan Deze methode is gemakkelijk te gebruiken en heeft het aantal verschillende relatief lage kosten van stappen, hoge reproduceerbaarheid De mogelijkheid om glycan isomers. De metingen van eiwitten in plasma kunnen aantrekkelijk zijn. in de geneeskunde.ook geassocieerd met Ig...

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

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

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

Behavior

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Biochemistry

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

Biology

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

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 ...

Bioengineering

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 ...

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

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