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1. Fluorescence Derivatization of Sialic Acids
<ol>
	<li>Prepare 10 mL of OPD-solution, consisting of 100 mg of o-phenylenediamine and 208 mg of sodium hydrogen sulfite in 10 mL of distilled&#160; H&#8322;O.</li>
	<li>Add 20 &#181;L of OPD-solution to the sialic acid samples. Vortex vigorously for 30 s and incubate samples for 4 h at 80 &#176;C in the dark (i.e. wrap the microtubes in aluminum foil).</li>
	<li>Let the samples cool down for 5 min, add 80 &#181;L of distilled&#160; H&#8322;O and centrifuge the tubes at 14,000 x g for 1 min.</li>
	<li>Transfer 80 &#181;L from the supernatant into a 300 &#181;L high-recovery HPLC vial. The derivatized sialic acid samples can be stored at 4-6 &#176;C for up to one week.</li>
</ol>
2. HPLC Analysis of Sialic Acid Derivatives
<ol>
	<li>Analyze the samples using a standard HPLC system connected to an online fluorescence detector.</li>
	<li>Use a reversed phase C18 column with the standard dimensions of 250 mm length and 4.6 mm diameter for the analysis.</li>
	<li>Prepare solvent A by diluting 200 mL of stock solution with 800 mL of LCMS-grade water (LCMS - liquid chromatography mass-spectrometry). The stock solution itself can be prepared as follows:
	<ol>
		<li>Add 46 g of formic acid to 800 mL of LCMS-grade H&#8322;O.</li>
		<li>Adjust the pH to 4.5 by dropwise adding ammonium hydroxide solution (puriss. p.a.).</li>
		<li>Transfer the solvent to a measuring cylinder and fill up to 1,000 mL with LCMS-grade&#160; H&#8322;O. This stock solution can be stored at 4-6 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>For solvent B, use LCMS-grade acetonitrile.</li>
	<li>Separate the sialic acids derivatives at a 1 mL/min flow rate with the following gradient elution:
	<ol>
		<li>Start by adding 10% of solvent B mixed to solvent A.</li>
		<li>From 0 min to 15 min, gradually increase the proportion of solvent B with a linear gradient to 60%.</li>
		<li>From 15 min to 16 min, rapidly increase the proportion of solvent B with a linear gradient from 60% to 90%. This initiates the washing of the HPLC column.</li>
		<li>To further wash the HPLC column, keep the level of solvent B at 90% between 16 min and 18 min before gradually reducing the level of solvent B again to 10% between 18 min and 19 min.</li>
		<li>Re-equilibrate HPLC column to the starting conditions of 10% B between 19 and 24 min.</li>
	</ol>
	</li>
	<li>Inject 50 &#181;L of sample into the HPLC system.</li>
	<li>Monitor eluents using the fluorescence detector excitation/emission wavelengths of 373/448 nm, and the derivatized sialic acids can be expected at the approximate retention times of 9 min (for Neu5Gc-OPD) and 10 min (for Neu5Ac-OPD).</li>
	<li>Calculate the relative amount of Neu5Gc (FNeu5Gc) from the fluorescence peak areas of the Neu5Ac (ANeu5Ac) and Neu5Gc (ANeu5Gc) as follows: 
	FNeu5Gc [%] = 100&#215;ANeu5Gc/(ANeu5Ac+ANeu5Gc). In case of the milk and liver samples from the homozygous Cmah knock-out mouse FNeu5Gc should be 0%, whereas the values for FNeu5Gc of heterozygous and wild-type mice may vary widely depending on mouse age and tissue type (between 2% and &#62;90%) with expected error margins of &#177;12%.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylneuraminic acid</td>
			<td>Sigma</td>
			<td>A0812</td>
			<td></td>
		</tr>
		<tr>
			<td>N-glycolylneuraminic acid</td>
			<td>Sigma</td>
			<td>50644</td>
			<td>1 mg aliquot should be sufficient</td>
		</tr>
		<tr>
			<td>O-phenylenediamine</td>
			<td>Sigma</td>
			<td>694975</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen sulfite</td>
			<td>J&#38;K Scientific Ltd</td>
			<td>75234</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>#5188-2788</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Column</td>
			<td>Phenomenex</td>
			<td>Hyperclone ODS</td>
			<td>250 x 4.6 mm</td>
		</tr>
		<tr>
			<td>LCMS-grade H2O</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr>
			<td>LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td>#100029</td>
			<td>Hypergrade</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Fluka</td>
			<td>#44273</td>
			<td>Puriss. p.a.</td>
		</tr>
	</tbody>
</table>

reversed-phase high-performance liquid chromatography: a robust method for quantitation of fluorescently labeled and derivatized sialic acids isolated from mouse liver

Source: Cao, C. et al. <a href="https://www.jove.com/v/56030">Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.</a>&#160;J. Vis. Exp.&#160;(2017)
In this video, we demonstrate the detection of sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, using high-performance liquid chromatography, HPLC, following derivatization with a suitable fluorescent labeling reagent to aid in fluorescence detection.

Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver

Source: Cao, C. et al. Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.&#160;J. Vis. Exp.&#160;(2017)
In this ...

Sialic acids, such as N-acetylneuraminic acid and N-glycolylneuraminic acid, are negatively charged carbohydrate moieties found at distal ends of oligosaccharide chains on mammalian cell surface.
To quantify relative amounts of these sialic acids in mouse liver tissue using reversed-phase high-performance liquid chromatography or RP-HPLC, first, treat the isolated sialic acid mixture with derivatization agent. The derivatization agent interacts with the functional groups of sialic acids, generating stable, fluorescent sialic acid derivatives.
Assemble an RP-HPLC column, connected to a fluorescence detector. The column comprises porous silica-based stationary phase bonded to non-polar, hydrophobic, alkyl chain ligands to facilitate selective interactions with sialic acid derivatives during chromatographic run.
Equilibrate the column using aqueous polar mobile phase containing low concentrations of acetonitrile, a less polar organic solvent, for optimal column conditioning.
Load the derivatized sialic acid mixture into the column.
N-acetylneuraminic acid, containing a hydrophobic N-acetyl group, adsorbs more strongly to the hydrophobic ligands of the stationary phase than N-glycolylneuraminic acid with a hydrophilic N-glycolyl group.

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Biological Techniques

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610 Views. Source: Cao, C. et al. Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. J. Vis. Exp. (2017) In this video, we demonstrate the detection of sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, using high-performance liquid chromatography, HPLC, following derivatization with a suitable fluorescent labeling reagent to aid in fluorescence detection. 

Video: Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver - Concept

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

JoVE Journal

Biochemistry

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Chemistry

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 ...

Reversed-Phase High-Performance Liquid Chromatography: A Robust Method for Quantitation of Fluorescently Labeled and Derivatized Sialic Acids Isolated from Mouse Liver

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