This work was supported by Iowa State University Startup Funds. F.E. was partially funded by the NSF Trinect Fellowship and Manley Hoppe Professorship. &#173;&#173;T.J.M. was partially supported by the Karen and Denny Vaughn Faculty Fellowship. The authors thank the Iowa State University Flow Cytometry Facility and the W.M. Keck Metabolomics Research Laboratory for assistance with fluorescence and LC-MS studies.

1. Cell culture and induction conditions
NOTE: In the following experiments, three strains are used: E. coli BL21 (DE3) with an empty vector, E. coli BL21 (DE3) with plasmids pAfcA14 and pET28:green fluorescent protein (GFP), and E. coli BL21 (DE3) with plasmids pAfcB14 and pET28:GFP. All strains are grown in Luria-Bertani broth (LB) or minimal media with appropriate antibiotics. Prepare stock solutions of 1,000x kanamycin (50 mg/mL) and carbenicillin (100 mg/mL) in deionized (DI) water.
<ol>
	<li>Media preparation
	<ol>
		<li>Prepare LB media by adding 25 g of LB powder stock to 1 L of DI water. Autoclave the solution at 121 &#176;C for 20 min to sterilize. Wait until the sterilized LB media temperature is below 60 &#176;C before addition of 1 &#181;L of antibiotic stock for every 1 mL of LB media.</li>
		<li>To prepare LB plates, add 15 g agar to the LB media before sterilization. Wait until the sterilized LB agar temperature is below 60 &#176;C before addition of antibiotic.</li>
		<li>1.1.3 For the biosensing cells, prepare media or plates with dual antibiotics, kanamycin and carbenicillin.</li>
	</ol>
	</li>
	<li>Carbohydrate inducers
	<ol>
		<li>Prepare stock solutions of the carbohydrate inducers at molar equivalents to 20 g/L of lactose: 29 g/L of 2&#8217;-FL and 3-FL. 
		NOTE: Each 200 &#181;L of cells will require 20 &#181;L of 2&#8217;-FL or 3-FL.</li>
		<li>Induce 200 &#181;L of cells with 2.0 g/L final concentration of lactose or 2.9 g/L final concentration of 2&#8217;-FL/3-FL. Incubate at 37 &#176;C with continuous shaking and let cultures grow overnight.</li>
	</ol>
	</li>
	<li>Cell culture
	<ol>
		<li>The day before the experiment seed 5 mL of LB medium supplemented with kanamycin and carbenicillin from a fresh bacterial colony, using sterile technique.</li>
		<li>Incubate all cultures at 37 &#176;C with agitation and grow the cultures overnight.</li>
		<li>Transfer 50 &#181;L of overnight culture into 5 mL of fresh LB/M9 media with kanamycin and carbenicillin. Incubate at 37 &#176;C with continuous shaking and grow until culture has reached optical density at 600 nm (OD600) of 0.5&#8211;0.7. At this mid-log phase, the cells can be induced.</li>
	</ol>
	</li>
</ol>
2. Measurement of fluorescence and limits of detection
<ol>
	<li>Preparation of cells for flow cytometry
	<ol>
		<li>Transfer the overnight cultures to 1.5 mL tubes and centrifuge at 12,500 x g for 1 min.</li>
		<li>Discard supernatant and re-suspend the pellet in 500 &#181;L of 1x phosphate-buffered saline (PBS; 6 mM Na2HPO4, 1.8 mM NaH2PO4, 145 mM NaCl in DI water, pH 7.2) to prepare a single cell suspension and transfer the single cell suspension to 5 mL flow cytometry tubes.</li>
		<li>Keep the cells in the dark at 4 &#176;C until running the samples on a flow cytometer. For best results, analyze the cells as soon as possible.</li>
		<li>Select 488 nm laser to excite GFP. Collect fluorescein isothiocyanate (FITC) fluorescence levels (20,000&#8211;40,000 events, gated for forward and side scatter to avoid aggregated cells and debris) with a 525/50 nm bandpass filter.</li>
	</ol>
	</li>
	<li>Calibrating the biosensor
	<ol>
		<li>To make a calibration curve, make 8&#8211;10 dilutions of the standard 2&#8217;-FL in the range 0&#8211;2,500 mg/L.</li>
		<li>Culture the 2&#8217;-FL biosensing cells (containing pAfcA and pET28:GFP) and induce them with the standard dilutions, as described previously in section 1.3. Carry out three biological replicates for each dilution.</li>
	</ol>
	</li>
</ol>
3. Detection and quantification of 2&#8217;-FL in a producer strain
<ol>
	<li>Cultivation of producer strain
	<ol>
		<li>For making minimal media, prepare separate solutions of 1 M K2HPO4, FeSO4&#183;7H2O, sodium citrate, and 1 M thiamine-HCl. Sterilize the solutions using a 0.22 &#181;m filter. Mix M9 media broth powder with 1 L of DI water. Autoclave the M9 solution at 121 &#176;C for 15 min. Cool the solution down to 50 &#176;C. Add MgSO4, CaCl2, K2HPO4, FeSO4&#183;7H2O, sodium citrate, and thiamine to final concentrations of 2 mM, 0.1 mM, 12 g/L, 11 mg/L, 75 mg/L, and 7.5 &#181;g/L, respectively.</li>
		<li>Start a culture of 2&#8217;-FL producing strain, e.g., JM109 gwBC-F2, in 5 mL of LB media and let it grow overnight at 37 &#176;C, as described in Baumgartner et al.12.</li>
		<li>Subculture at 1% as described earlier in step 1.3.3 in 250 mL of the minimal media prepared in step 3.1.1. Add glycerol to a final concentration of 10 g/L and incubate the culture at 37 &#176;C.</li>
		<li>When the culture reaches an OD600 of 0.5&#8211;0.7, add isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and lactose to a final concentration of 2 g/L.</li>
		<li>Incubate at 37 &#176;C with continuous shaking and let cultures grow for 24 h.</li>
	</ol>
	</li>
	<li>Extraction of 2&#8217;-FL
	<ol>
		<li>Transfer the overnight cultures to sterile 50 mL tubes and centrifuge at 900 x g for 15 min.</li>
		<li>3.2.2 Discard supernatant and re-suspend the pellet in DI water. Repeat the wash step three times to remove residual lactose and finally re-suspend in 5 mL of DI water.</li>
		<li>To lyse the cell suspension, use a sonicator at 30% power, in 30 s pulses. Keep the cells on ice at all times.</li>
		<li>Centrifuge the lysed cells for 25 min at 2,000 x g. Remove the supernatant, pass it through a sterile (e.g., 0.22 &#181;m) filter and store it at 4 &#176;C until analysis.</li>
	</ol>
	</li>
	<li>Quantification with biosensor
	<ol>
		<li>Inoculate a culture of 2&#8217;-FL biosensing cells and at mid-log phase, induce with cell lysate equivalent to estimated concentrations of 2&#8217;-FL used to make the calibration curve as described in section 2.2. Perform the calibration in the same milieu as the sample to be analyzed (e.g., cell lysate) by adding dilutions of 2&#8217;-FL to control (i.e., non-producer) filtered cell lysate before inducing biosensor cells.</li>
		<li>Run the samples on a flow cytometer. Generate a dose-response curve by plotting the fluorescence output against the oligosaccharide concentration. Compare the response of the standards to the cell lysate.</li>
	</ol>
	</li>
</ol>
4. Selective removal of lactose
NOTE: The biosensing cells are sensitive to lactose and it is desirable for the biosensor to selectively detect HMOs over lactose. One strategy would include washing of cells as described in section 3.2. Three other strategies are described below.
<ol>
	<li>Treatment with commercial purified &#946;-galactosidase
	<ol>
		<li>Dissolve lyophilized &#946;-galactosidase to 1,000 (FCC lactase) units/mL, in 1 mM MgCl2 or use commercial &#946;-galactosidase in solution.</li>
		<li>To determine the optimum concentration of enzyme needed, grow an inoculum of 2&#8217;-FL biosensing cells and induce with 2 g/L lactose and 2.9 g/L 2&#8217;-FL at mid log phase.</li>
		<li>To 100 &#181;L cultures, add variable amounts of &#946;-galactosidase, up to 12 units, and let the cultures grow overnight at 37 &#176;C with continuous shaking.</li>
		<li>Measure the fluorescence as described in section 2.1 and calculate the optimum units of enzyme needed by determining the minimum enzyme concentration to achieve desired attenuation of signal.</li>
		<li>To 2&#8217;-FL producing cells grown for 24 h, add optimum units of &#946;-galactosidase and incubate overnight at 37 &#176;C. Proceed with the protocol for determining the 2&#8217;-FL titer as described in section 3.3.</li>
	</ol>
	</li>
	<li>Pre-treatment with LacZ+ strain
	<ol>
		<li>Start a 25 mL culture of 2&#8217;-FL producing strain and once induced at mid-log phase, incubate the culture at 37 &#176;C for 24 h.</li>
		<li>Inoculate an E. coli BL21 (DE3) culture in LB, grow it to mid-log phase at 37 &#176;C and add 50 mL of the culture (2:1) to the 24 h culture.</li>
		<li>Incubate at 37 &#176;C for 3 h. Proceed with the protocol for determining the 2&#8217;-FL titer as described in section 3.3.</li>
	</ol>
	</li>
	<li>Lactose precipitation with ethanol 
	NOTE: This section is adapted from Matsuki et al.12.
	<ol>
		<li>Start a 25 mL culture of 2&#8217;-FL producing strain and once induced at mid-log phase, incubate at 37 &#176;C for 24 h.</li>
		<li>Add two volumes of 100% ethanol to the culture, shake well, and incubate at 37 &#176;C for 4 h.</li>
		<li>Centrifuge the suspension at 900 x g for 15 min. Collect the supernatant and incubate overnight at 4 &#176;C, which precipitates the lactose.</li>
		<li>Remove the precipitated lactose by passing the solution through a filter paper (11 &#181;m). Collect the filtrate that is the cell lysate containing the 2&#8217;-FL, which can be quantified by following section 3.3.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We present a high-throughput strategy for the linkage-specific detection of fucosylated human milk oligosaccharides. This was accomplished by building whole cell biosensors by genetically engineering E. coli which upon induction with specific glycans respond with a fluorescent signal. The protocol also details on how the biosensor can be used to detect and quantify HMOs in a metabolically engineered bacterial strain.
Our protocol offers advantages over alternative methods due to its h...

Human milk oligosaccharides (HMOs) are lactose-derived oligosaccharides, usually comprising three to eight sugar monomers. They have a lactose (Gal-&#946;1,4-Glc) reducing end and are further elongated by glycosidic links (&#946;-1,3- or &#946;-1,6-) to glucose (Glc), galactose (Gal), or N-acetylglucosamine (GlcNAc). In addition, fucose (Fuc, &#945;-1,2- or &#945;-1,3-) or sialic acid (Sia or NeuAc, &#945;-2,3- or &#945;-2,6-) residues are often added1.
Current analysis of oligosaccharides and other carbohydrates is limited in throughput and scope by the need for chromatographic/mass spectrometric (MS) technology2,3,4,5,6,7, which can take roughly an hour per sample, not to mention the necessity for expensive equipment, specialized columns and derivatizing agents, and expertise on the operation of this equipment8. Oligosaccharide linkages are particularly difficult to determine, requiring advanced MS9,10 or nuclear magnetic resonance (NMR) techniques11. Rapid optimization of synthesis of these oligosaccharides is thus limited by the throughput of this slow analytical step.
In this study, we demonstrate linkage-specific detection of fucosylated trisaccharide lactose-based HMOs, focusing on 2&#8217;-fucosyllactose (2&#8217;-FL) that is the most abundant HMO in human milk, using a genetically encoded Escherichia coli whole cell biosensor with a limit of detection at 4 mg/L. An important feature of this biosensor is its ability to distinguish between isomeric trisaccharides. The design principle is based on the expression of specific fucosidases in E. coli that liberate lactose from HMOs, the presence of which is detected by the lac operon, which in turn generates a fluorescent signal. We achieve this by building a two-plasmid system, one harboring the linkage-specific fucosidase and the other a fluorescent reporter protein. This biosensor platform is suitable for high-throughput screening by flow cytometry or micro-plate reader. We also demonstrate the utilization of the biosensor in quantifying 2&#8217;-FL produced by an engineered strain12. Within this study, we also present three strategies on selective removal of lactose that can result in false positive signal from the biosensor, given that the engineered producer strain is grown on lactose.
Taken together, the genetically encoded biosensors allow us to detect and quantify HMOs in a linkage-specific manner, which is difficult even with chromatographic, MS, or NMR techniques. Due to its high throughput and ease of use, this method should have widespread applications in the metabolic engineering and synthesis of HMOs.

<table><tbody><tr><td>2&amp;rsquo;-Fucosyllactose&amp;nbsp;</td><td>Carbosynth&amp;nbsp;</td><td>41263-94-9</td><td></td></tr><tr><td>3-Fucosyllactose&amp;nbsp;</td><td>Carbosynth&amp;nbsp;</td><td>41312-47-4</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP9744500</td><td></td></tr><tr><td>Calcium Chloride, Dihydrate</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Carbenicillin&amp;nbsp;</td><td>Fisher Scientific</td><td>BP26481</td><td></td></tr><tr><td>Dextrose (D-Glucose), Anhydrous</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Flow Cytometer</td><td>BD</td><td></td><td>FACSCanto Plus RUO</td></tr><tr><td>HPLC</td><td>Agilent Technologies</td><td></td><td>1100 Series HPLC system</td></tr><tr><td>HPLC Column</td><td>Luna</td><td></td><td>C18 reversed phase column</td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>11815024</td><td></td></tr><tr><td>LB Broth, Miller&amp;nbsp;</td><td>Fisher Scientific</td><td>12-795-027</td><td></td></tr><tr><td>Lactose</td><td>Fisher Scientific</td><td>64044-51-5</td><td></td></tr><tr><td>M9, Minimimal Salts, 5x</td><td>Sigma-Aldrich</td><td>M6030</td><td></td></tr><tr><td>Magnesium Sulfate, Anhydrous</td><td>Fisher Scientific</td><td>M65-500</td><td></td></tr><tr><td>MS</td><td>Agilent Technologies</td><td></td><td>Mass Selective Trap SL detector</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>7558-79-4</td><td></td></tr><tr><td>Sodium phosphate monobasic</td><td>Sigma-Aldrich</td><td>13472-35-0</td><td></td></tr></tbody></table>

analysis of fucosylated human milk trisaccharides in biotechnological context using genetically encoded biosensors

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of detection and quantification of monosaccharide and linkages. Conventional techniques of glycan analysis include chromatographic/mass-spectrometric methods with throughput on the order of hundreds of samples per day without automation. We demonstrate here, a genetically encoded bacterial biosensor for the high-throughput, linkage-specific detection and quantification of the fucosylated HMO structures, 2&#8217;-fucosyllactose and 3-fucosyllactose, which we achieved via heterologous expression of fucosidases. As the presence of lactose in milk or in biotechnological processes could lead to false positives, we also demonstrate the reduction of signal from lactose using different strategies. Due to the high throughput of this technique, many reaction conditions or bioreactor parameters could be assayed in parallel in a matter of hours, allowing for the optimization of HMO manufacturing.

We designed a whole cell biosensor specific to 2&#8217;-FL that can be used in conjunction with the biotechnological production of the oligosaccharide. This relies on the specific enzymatic cleavage of modifying terminal sugars generating lactose and thereby activation of the lac operon, leading to expression of a reporter fluorescent protein under a lactose inducible promoter, in proportion to the quantity of 2&#8217;-FL. To demonstrate its linkage specificity, 3-fucosyllactose ...

Watch this Scientific Journal Video about Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors at JoVE.com

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

We describe here the high-throughput detection and quantification of fucosylated human milk oligosaccharides (HMOs) using a whole-cell biosensor. We also demonstrate here, the adaptation of this platform towards analysis of HMO production strains, focusing on improving the signal to noise ratio.

Human milk oligosaccharides (HMOs) are complex carbohydrate components of human breast milk that exhibit plentiful benefits on infant health. However, ...

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Biochemistry

6.2K Views.  Iowa State University. Deze methode kan helpen bij het beantwoorden van een aantal belangrijke vragen die betrokken zijn bij de productie van menselijke melk oligosaccharides. Ten eerste, hoeveel van het product heb ik? En ten tweede, wat is de specifieke koolhydraat koppeling?Het doel hier is om het proces van kwantificeren en karakteriseren van deze menselijke melk oligosadariden te vereenvoudigen en te versnellen. Deze methode heeft twee voordelen ten opzichte van de huidige stand van de techniek. Ten eer...

Video: Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

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

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Bioengineering

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Biology

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 ...

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

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

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.

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.

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

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 ...

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 ...

Analysis of Fucosylated Human Milk Trisaccharides in Biotechnological Context Using Genetically Encoded Biosensors

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