Name: glycan node analysis: a bottom-up approach to glycomics
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Description: Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

This work was supported by the College of Liberal Arts and Sciences of Arizona State University in the form of laboratory startup funds to CRB. It was also supported by a grant from Flinn Foundation (Grant No. 1977) and by the National Cancer Institute of the National Institutes of Health under Award Number R33CA191110. JA was supported by the National Institute of General Medical Sciences of the National Institutes of Health Postbaccalalureate Research Education Program (PREP) under award number R25GM071798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Caution: Avoid skin/eye contact with any of the reagents used in this experiment. Upon exposure, thoroughly flush the affected area with water and seek immediate medical advice.
1. Permethylation and Glycan Extraction
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	<li>Column Preparation
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		<li>Obtain as many microfuge spin column units as the samples to be analyzed. Break and detach the plastic reservoir tube caps. Place a microfuge mini filter in each reservoir tube. Place the assembled microfuge spin columns (co...

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	<li>Li, M., Song, L., Qin, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+changes:+cancer+metastasis+and+anti-cancer+vaccines.">Glycan changes: cancer metastasis and anti-cancer vaccines.</a> J Biosciences. 35 (4), 665-673 (2010).</li><li>Stanley, P., Schachter, H., Taniguchi, N., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , Ch. 8: N-Glycans (2009).</li><li>Apweiler, R., Hermjakob, H., Sharon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+frequency+of+protein+glycosylation,+as+deduced+from+analysis+of+the+SWISS-PROT+database.">On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.</a> Biochim Biophys Acta. 1473 (1), 4-8 (1999).</li><li>Brockhausen, I., Schachter, H., Stanley, P., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Rini, J., Esko, J., Varki, A., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Gercel-Taylor, C., Bazzett, L. B., Taylor, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+aberrant+tumor-reactive+immunoglobulins+in+the+circulation+of+patients+with+ovarian+cancer.">Presence of aberrant tumor-reactive immunoglobulins in the circulation of patients with ovarian cancer.</a> Gynecol Oncol. 81 (1), 71-76 (2001).</li><li>An, H. 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=Profiling+of+glycans+in+serum+for+the+discovery+of+potential+biomarkers+for+ovarian+cancer.">Profiling of glycans in serum for the discovery of potential biomarkers for ovarian cancer.</a> J Proteome Res. 5 (7), 1626-1635 (2006).</li><li>Kanoh, 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=Changes+in+serum+IgG+oligosaccharide+chains+with+prostate+cancer+progression.">Changes in serum IgG oligosaccharide chains with prostate cancer progression.</a> Anticancer Res. 24 (5B), 3135-3139 (2004).</li><li>Kyselova, Z., 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=Alterations+in+the+serum+glycome+due+to+metastatic+prostate+cancer.">Alterations in the serum glycome due to metastatic prostate cancer.</a> J Proteome Res. 6 (5), 1822-1832 (2007).</li><li>Okuyama, N., 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=Fucosylated+haptoglobin+is+a+novel+marker+for+pancreatic+cancer:+a+detailed+analysis+of+the+oligosaccharide+structure+and+a+possible+mechanism+for+fucosylation.">Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation.</a> Int J Cancer. 118 (11), 2803-2808 (2006).</li><li>Zhao, 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=Glycoprotein+microarrays+with+multi-lectin+detection:+unique+lectin+binding+patterns+as+a+tool+for+classifying+normal,+chronic+pancreatitis+and+pancreatic+cancer+sera.">Glycoprotein microarrays with multi-lectin detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera.</a> J Proteome Res. 6 (5), 1864-1874 (2007).</li><li>Comunale, M. 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N., 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=Novel+glycan+biomarkers+for+the+detection+of+lung+cancer.">Novel glycan biomarkers for the detection of lung cancer.</a> J Proteome Res. 10 (4), 1755-1764 (2011).</li><li>Bones, J., Mittermayr, S., O'Donoghue, N., Guttman, A., 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=Ultra+performance+liquid+chromatographic+profiling+of+serum+N-glycans+for+fast+and+efficient+identification+of+cancer+associated+alterations+in+glycosylation.">Ultra performance liquid chromatographic profiling of serum N-glycans for fast and efficient identification of cancer associated alterations in glycosylation.</a> Anal Chem. 82 (24), 10208-10215 (2010).</li><li>Kodar, K., Stadlmann, J., Klaamas, K., Sergeyev, B., Kurtenkov, O. <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+Fc+N-glycan+profiling+in+patients+with+gastric+cancer+by+LC-ESI-MS:+relation+to+tumor+progression+and+survival.">Immunoglobulin G Fc N-glycan profiling in patients with gastric cancer by LC-ESI-MS: relation to tumor progression and survival.</a> Glycoconj J. 29 (1), 57-66 (2012).</li><li>Chen, G., 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=Human+IgG+Fc-glycosylation+profiling+reveals+associations+with+age,+sex,+female+sex+hormones+and+thyroid+cancer.">Human IgG Fc-glycosylation profiling reveals associations with age, sex, female sex hormones and thyroid cancer.</a> J Proteomics. 75 (10), 2824-2834 (2012).</li><li>Takeda, 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=Fucosylated+haptoglobin+is+a+novel+type+of+cancer+biomarker+linked+to+the+prognosis+after+an+operation+in+colorectal+cancer.">Fucosylated haptoglobin is a novel type of cancer biomarker linked to the prognosis after an operation in colorectal cancer.</a> Cancer. 118 (12), 3036-3043 (2012).</li><li>Parekh, R. 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S., 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=Increased+levels+of+galactose-deficient+anti-Gal+immunoglobulin+G+in+the+sera+of+hepatitis+C+virus-infected+individuals+with+fibrosis+and+cirrhosis.">Increased levels of galactose-deficient anti-Gal immunoglobulin G in the sera of hepatitis C virus-infected individuals with fibrosis and cirrhosis.</a> J Virol. 82 (3), 1259-1270 (2008).</li><li>Horvat, T., Zoldo&#353;, V., Lauc, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutional+and+clinical+implications+of+the+epigenetic+regulation+of+protein+glycosylation.">Evolutional and clinical implications of the epigenetic regulation of protein glycosylation.</a> Clinical Epigenetics. 2 (2), 425-432 (2011).</li><li>Zoldo&#353;, V., Novokmet, M., Be&#269;eheli, I., Lauc, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomics+and+epigenomics+of+the+human+glycome.">Genomics and epigenomics of the human glycome.</a> Glycoconj J. 30 (1), 41-50 (2013).</li><li>Lowe, J. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+cellular+mechanisms+of+health+and+disease.">Glycosylation in cellular mechanisms of health and disease.</a> Cell. 126 (5), 855-867 (2006).</li><li>Bertozzi, C. R., Sasisekharan, R., Varki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2009).</li><li>Borges, C. R., Rehder, D. S., Boffetta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+surrogate+analysis+of+glycotransferase+activity+in+whole+biospecimens.">Multiplexed surrogate analysis of glycotransferase activity in whole biospecimens.</a> Anal Chem. 85 (5), 2927-2936 (2013).</li><li>Mcinnes, A. G., Ball, D. H., Cooper, F. P., Bishop, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+Carbohydrate+Derivatives+by+Gas-Liquid+Partition+Chromatography.">Separation of Carbohydrate Derivatives by Gas-Liquid Partition Chromatography.</a> J Chromatogr. 1 (6), 556-557 (1958).</li><li>Ciucanu, I., Caprita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Per-O-methylation+of+neutral+carbohydrates+directly+from+aqueous+samples+for+gas+chromatography+and+mass+spectrometry+analysis.">Per-O-methylation of neutral carbohydrates directly from aqueous samples for gas chromatography and mass spectrometry analysis.</a> Anal Chim Acta. 585 (1), 81-85 (2007).</li><li>Ciucanu, I., Kerek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+the+permethylation+of+carbohydrates.">A simple and rapid method for the permethylation of carbohydrates.</a> Carbohydr Res. 131, 209-217 (1984).</li><li>Kang, P., Mechref, Y., Klouckova, I., Novotny, M. 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In general, the successful production of partially methylated alditol acetates (PMAAs) from hexoses is fraught with fewer difficulties and is more robust than the successful production of N-acetylhexosamine (HexNAc) PMAAs. The exact mechanism behind this phenomenon as it plays out in every step of this procedure is unknown, but must relate to the unique chemistry of the N-acetyl group (rather than hydroxyl group) that is unique to HexNAcs relative to hexoses. The mechanism behind this phenomenon as it r...

Glycolipids, glycoproteins, proteoglycans, and glycosaminoglycans constitute the four main classes of complex, heterogeneous carbohydrates collectively known as glycans. As ubiquitous and integral components of the plasma membrane, glycocalyx, and extracellular matrix and fluids, glycans partake in such diverse biochemical processes as endocytosis, intracellular trafficking, cell motility, signal transduction, molecular recognition, receptor activation, cell adhesion, host-pathogen interaction, intercellular communication, immunosurveillance, and immune response initiation.1 Present in nearly every domain of life, enzymes known as glycosyltransferases that build glycan polymers act in tandem with glycoside hydrolases (also known as glycosidases, which break down glycans) to construct, remodel, and ultimately produce finalized glycan polymers 2. Although each glycosyltransferase may operate on different glycoconjugates, a glycosyltransferase generally forges a linkage- and anomer-specific glycosidic bond by transferring the monosaccharide moiety of a particular activated nucleotide sugar donor (e.g., GDP-fucose) to a certain category of nucleophilic acceptors (e.g., a lipid, polypeptide, nucleic acid, or growing oligosaccharide). It has been estimated that more than 50% of proteins (especially membrane and secretory proteins) are post-translationally modified by glycosylation.3 Rudimentary combinatorial calculations provide an appreciation for the considerable variability, versatility, and specificity accorded to glycoproteins by glycosylation; for example, if a polypeptide substrate has only 10 glycosylation sites and each site can form a glycosidic linkage with 1 of only 3 different monosaccharide reducing ends, then, theoretically, the final glycoprotein can assume 310 = 59,049 distinct identities. In glycoproteins, glycosidic linkages commonly form with the side-chain nitrogen of asparagine residues in the sequence Asn-X-Ser/Thr (X can be any amino acid except proline) to yield N-glycans2 and side-chain hydroxyls of serine and threonine residues to yield O-glycans4. The composition of a cell&#39;s glycome (i.e., its complement of glycosylation products) is unique and limited because, with few exceptions, glycosyltransferases exhibit strict donor, acceptor, and linkage specificity.5 Important and abundant blood plasma glycoproteins suffer aberrant glycosylation as a downstream consequence of abnormal glycosyltransferase expression and activity due to many pathological conditions, especially cancer and inflammatory diseases.6-24
Mainly due to epigenetic factors, the glycome is significantly more diverse, dynamic, and complex than the proteome and transcriptome.25,26 While approximately 1% of the mammalian genome encodes the formation, modification, and assembly of glycans,27 glycosylation proceeds in a non-template-driven manner-a marked contrast to polypeptide and nucleic acid biosynthesis. The interplay among the relative quantity and activity of glycosylation enzymes and such environmental factors as nutrient and precursor availability ultimately determines the nature, rate, and extent of glycosylation.5,28 Embryogenesis (e.g., determination and differentiation), cellular activation, and progression through the cell cycle influence gene expression (i.e., transcription and translation) and alter the identity and quantity of available glycosyltransferases, whose activity is the immediate upstream determinant of the cell&#39;s glycan profile. Because (some of) the proliferative, adhesive, and invasive properties of cancerous cells resemble those of ordinary embryogenic cells, specific changes in glycan biosynthetic pathways (e.g., precursor accumulation, deregulated expression, aberrant modification, structural truncation, or novel formation) serve as universal cancer biomarkers that indicate various stages of tumor formation, progression, migration, and invasion.29 Although glycosylation is highly complex, evidently only a few alterations in glycosylation can enable carcinogenesis and metastasis; apparently, certain &#34;aberrant&#34; glycosylation products indeed benefit cancerous cells by enabling them to evade immune recognition and survive the demands of migration in inhospitable intravascular and metastatic environments.28,30,31 Not surprisingly, experiments have revealed that disrupting or preventing patterns of altered gene expression and aberrant glycan formation can halt tumorigenesis.29 Nonetheless, the aberrant glycans detected in a biofluid sample (e.g., urine, saliva, and blood plasma or serum) may not be direct indicators of cancer (or another disease), but rather downstream outcomes of subtle yet significant changes in the immune system or quantifiable ramifications of a pernicious condition in an unpredictable organ.32
Although they provide universal information about the glycome, many molecular interaction-based glycomics techniques (e.g., lectin/antibody arrays and metabolic/covalent labeling) depend upon the detection of whole glycan structures and do not provide detailed structural information about individual glycans. In marked contrast, mass spectrometry (MS) can help identify and quantify individual glycan structures and reveal such structural information as the attachment sites to polypeptide cores. Deregulated expression or activity of only one glycosyltransferase can initiate a cascade of detrimental molecular events in multiple glycosylation pathways. Because each glycosyltransferase may operate on more than one glycoconjugate substrate and across different growing glycan polymers, deregulated biosynthetic cascades yield disproportionally increased amounts of only one glycan product but several heterogeneous classes of aberrant glycans in intra- or extracellular fluids.33 However, such unique aberrant glycans are sometimes considered impractical as biomarkers for cancer or other glycan-affective pathologies because, compared to the large pool of well-regulated glycans, these aberrant glycans represent a very small fraction that may often remain undetectable even by such highly sensitive techniques as mass spectrometry. For example, in intra- and extracellular body fluids, the broad protein-concentration spectrum (which spans eight orders of magnitude) can prevent detection of scarce glycoproteins that are masked by the more abundant species. 32 Moreover, determining glycosyltransferase activity remains a considerable practical and theoretical challenge because many glycosyltransferases are absent in clinical biofluids or become inactive ex vivo. Despite the difficulty of consistently detecting and quantifying ultra-minute quantities of unique whole glycans, practitioners of mass spectrometry have made enormous strides toward employing intact glycans as clinical markers. We have recently developed a complementary approach to the analysis of intact glycans that, employing GC-MS, facilitates the detection of all constituent branch-point and linkage-specific monosaccharides (&#34;glycan nodes&#34;) that together impart uniqueness to each glycan and in many cases directly serve as molecular surrogates that quantify the relative activity of the culpable glycosyltransferase(s).
Since its first reported direct application to glycan analysis in 1958, gas chromatography (GC) has proven a powerful technique to analyze per-methylated mono- and disaccharides,34 determine their anomericity and absolute configuration, and separate them for subsequent mass spectrometric analysis.35 Between 1984 and 2007, Ciucanu and colleagues introduced and refined a solid-phase glycan permethylation technique that employed sodium hydroxide and iodomethane, followed by liquid/liquid extraction of permethylated glycans using water and chloroform.35,36 Between 2005 and 2008, Kang and co-workers integrated a time-saving spin-column approach into the permethylation step.37,38 In 2008, the Goetz research group devised a quantitative solid-phase permethylation glycan-profiling method using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to compare and potentially distinguish invasive and non-invasive breast cancer cells;39 then, in 2009, the Goetz team combined enzymatic and chemical release techniques to cleave O-glycans from intact glycoproteins in a highly alkaline solid-phase permethylation scheme.40 Although the Goetz procedure facilitated simultaneous permethylation and chemical release of O-glycans, it was applied only to pre-isolated glycoproteins. We modified this technique in 2013 and adapted it for whole unfractionated biofluids and homogenized tissue samples by incorporating trifluoroacetic acid (TFA) hydrolysis, reduction, and acetylation steps.33 These additional steps also release glycans from glycolipids and N-linked glycans from glycoproteins and convert them into partially methylated alditol acetates (PMAAs, Figure 1), whose distinctive methylation-and-acetylation patterns facilitate analysis by GC-MS and uniquely characterize the constituent glycan nodes in the original intact glycan polymer41 (Figure 2).33 Ultimately, this procedure produces a composite portrait of all the glycans in a complex biofluid based on direct, relative quantification of unique glycan features such as &#34;core fucosylation&#34;, &#34;6-sialylation&#34;, &#34;bisecting GlcNAc&#34;, and &#34;beta 1-6 branching&#34;-each derived from a single GC-MS chromatographic peak. This article presents further optimization of the permethylation, acetylation, isolation, and clean-up stages along with improvements in the mode of relative quantification.

<table>
	<tbody>
		<tr>
			<td>Sodium hydroxide beads&#160;</td>
			<td>367176</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>20-40 mesh, reagent grade 97%</td>
		</tr>
		<tr>
			<td>0.9 mL Spin column</td>
			<td>69705</td>
			<td>Pierce division of ThermoFisher Scientific&#160;</td>
			<td>Includes&#160; plugs and polyethylene frits&#160;</td>
		</tr>
		<tr>
			<td>GC-MS autosampler vial (silanized)*</td>
			<td>C4000-9</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Target DP High Recovery Vial, 1.5 mL, 12 x 32 mm, Includes Teflon-lined pierceable caps</td>
		</tr>
		<tr>
			<td>1.5 mL polypropylene test tubes</td>
			<td>05-402-25</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Snap-cap lid</td>
		</tr>
		<tr>
			<td>2 mL polypropylene test tubes</td>
			<td>05-408-138</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Snap-cap lid</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D8418</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>BioReagent for molecular biology, reagent grade &#62;99.0%</td>
		</tr>
		<tr>
			<td>Iodomethane&#160;</td>
			<td>I8507</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Contains copper as stabilizer, ReagentPlus 99%</td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>A955-4</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Optima LC/MS</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>75002436: Sorvall Legend Micro 17 Centrifuge</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>24 x 1.5/2.0 rotor with ClickSeal biocontainment lid. Rotor catalog number: 75003424</td>
		</tr>
		<tr>
			<td>13 x 100 glass test tube (silanized)*</td>
			<td>53283-800</td>
			<td>VWR</td>
			<td>13 x 100 mm borosilicate glass test tubes with screw-cap finish</td>
		</tr>
		<tr>
			<td>Caps for glass test tubes</td>
			<td>14-930-15D</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Kimble&#8482; Black Phenolic Screw Caps; 13mm-415 GPI thread; PTFE-faced rubber liner.</td>
		</tr>
		<tr>
			<td>Sodium chloride&#160;</td>
			<td>S7653</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#62;99.5% pure</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>4440-08</td>
			<td>Macron Fine Chemicals&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid&#160;</td>
			<td>299537</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99% purified by redistillation for protein sequencing&#160;</td>
		</tr>
		<tr>
			<td>Sodium borohydride</td>
			<td>71321</td>
			<td>Fluka Analytical&#160;</td>
			<td>99%</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution&#160;</td>
			<td>320145</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>NH3 content: 28.0 - 30.0%</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>AH230-4</td>
			<td>Honeywell Burdick &#38; Jackson</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>320099</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99.70%</td>
		</tr>
		<tr>
			<td>Plastic vacuum desiccator&#160;</td>
			<td>Any model of adequate size</td>
			<td>FoodSaver</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic anhydride&#160;</td>
			<td>539996</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>99.50%</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>D143SK-4</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Stabilized HPLC grade</td>
		</tr>
		<tr>
			<td>Acetone&#160;</td>
			<td>9006-03</td>
			<td>J.T.Baker</td>
			<td>Baker Analyzed&#160;</td>
		</tr>
		<tr>
			<td>Heated evaporation manifold (main unit)</td>
			<td>pi18823</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Thermo Scientific* Reacti-Therm* Heating and Stirring Module; Triple-block Model with Heating and Stirring Function</td>
		</tr>
		<tr>
			<td>Heated evaporation manifold (overhead evaporator)</td>
			<td>pi18826</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>ThermoScientific* Reacti-Vap Evaporator, 27-port; For use with triple-block Reacti-Therm heating module</td>
		</tr>
		<tr>
			<td>Aluminum sample-holder blocks for evaporation manifold</td>
			<td>pi18816</td>
			<td>ThermoFisher Scientific&#160;</td>
			<td>Block, Aluminum, Reacti-Block S-1, Holds 13mm dia test tubes, 13 holes (14 dia. x 45mm deep)</td>
		</tr>
		<tr>
			<td>Gas chromatograph</td>
			<td>Model A7890</td>
			<td>Agilent&#160;</td>
			<td>Equipped with CTC PAL autosampler</td>
		</tr>
		<tr>
			<td>Mass spectrometer&#160;</td>
			<td>GCT Premier (Time-of-Flight)</td>
			<td>Waters&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Split-mode liner (deactivated / silanized)</td>
			<td>5183-4647</td>
			<td>Agilent</td>
			<td>Containing a small plug of silanized glass wool</td>
		</tr>
		<tr>
			<td>DB-5ms GC column</td>
			<td>122-5532</td>
			<td>Agilent</td>
			<td>30 m x 0.25 mm ID x 0.25 micron film thickness</td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane</td>
			<td>95541</td>
			<td>Sigma-Aldrich&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vacuum desiccator (for glassware silanization)</td>
			<td>EW-06536-30</td>
			<td>Cole-Parmer</td>
			<td>12&#34; wide; 230 mm plate size</td>
		</tr>
		<tr>
			<td colspan="4">*Glassware silanization is carried out in-house, overnight using chlorotrimethylsilane vapor in a large glass vacuum desiccator.</td>
		</tr>
	</tbody>
</table>

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.

A total ion current chromatogram (TIC) showing successful permethylation, hydrolysis, reduction, and acetylation of human blood plasma samples relative to cases in which two critical permethylation steps were executed incorrectly are shown in Figure 3.
Absolute Yield of HexNAcs Relative to Hexoses:
N-acetylhexosamine (HexNAc) partiall...

Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

Glycan Node Analysis: A Bottom-up Approach to 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 ...

The overall goal of this procedure is to break down all glycans in a biological sample while conserving monosaccharide composition and linkage information. Facilitating distillation of interesting glycan features such as sick cyalalation and bisecting N-Acetylglucosamine into single analytical signals. This method can help to directly answer key questions in the field of glycomites such as which unique glycan features are altered in particular disease states.The main advantage of this technique is that glycan features that tend to be altered in disease such as, beta 1-6 branching and corfulcosulation are distilled into single analytical signals rather than being spread across multiple intact glycans. Generally, individuals need to do this method really struggle with poor yeilds of N-Acetylcysteines due to improper processing of freshly permethylated glycans. Prep the samples, by first transferring nine micro-liters of each biological analyte into separate 1.5 milliliter test tubes.To each sample, add one micro-liter of deionized water and 270 micro-liters of high purity of DMSO. And vigorously mix by vortexing them. After sample preparation, assemble the sodium hydroxide loaded spin columns as described in the text protocol.Using and analytical syringe, add 105 micro-liters of iodomethane to each analyte and immediately cap the tubes. Thoroughly vortex the samples. Unplug the previous prepared spin columns and centrifuge them for 15 seconds at 2, 400 Gs.After discarding the reservoir tubes and affluent, re-plug the columns and place them inside of a fresh set of tubes, labeled for each analyte.Use a 1000 micro-liter pipe head to transfer each analyte solution onto the corresponding spin column. Once the analyte solution is added, crimp about two milliliters from the end of a 200 micro-liter pipe head tip and leave the tip in the column to use as a stirrer. Use the pipe head tips to gently stir the columns once ever two to three minutes.After 11 minutes, unplug the columns and centrifuge them for 15 seconds at 2, 400 Gs.Immediately remove the spin columns and place them in new reservoir tubes, saving the flow-through solution but discarding the plugs. To the columns, add 300 micro-liters of acetonenitrile and centrifuge them 30 seconds at 9, 600 Gs.Now transfer the first permethylated flow-through solutions into silanized glass test tubes containing 3.5 milliliters of sodium phosphate buffer. Be sure that the transferred supernaned is clear and that no solid material is transferred.A small white pellet will have formed at the bottom of the plastic tubes. Avoid transferring any obvious solid material. Failure to do so will result poor yields of N-Acetylcysteine-glucosamine glycan notes.Immediately vortex every sample in the batch once all samples have been transferred. Then combine the acetonenitrile flow-through solutions with the rest of the liquid in the respective test tubes. Again, avoid transferring any solid, white residue during this step.Cap, shake and vortex the combined solutions immediately before moving to the next sample. After permethylation, add 1.2 milliliters of chloroform to each test tube inside a fume hood. Then cap the tubes and mix the contents vigorously.To separate the layers, briefly centrifuge the solutions at 3, 000 Gs.Use a pasture pipe head to extract and discard most of the top aqueous layer leaving about three milliliters of the layer behind. Add 3.5 milliliters of the 0.2 molar sodium phosphate buffer and then cap, mix and centrifuge the test tube. Execute this liquid/liquid extraction step a total of three times.Leaving about one milliliter of the aqueous layer in the final extraction. Now use a clean silanized pasture pipe head to carefully transfer the chloroform layer to a clean and labeled silanized glass test tube as described in the text protocol. In a pre-heated evaporation manifold, set to 75 degrees celsius, dry the samples under a gentle stream of nitrogen gas.Make sure that the gas flow disturbs the liquid surface but without splattering it. Once the samples are dry, they are ready for hydrolysis. Add 325 micro-liters of 2 Molar trifluoro acetic acid or TFA to each sample and mix well.Tightly cap each sample tube immediately after addition to all the samples to prevent TFA evaporation and sample loss during the subsequent heating step. Incubate the samples at 121 degrees celsius for two hours to hydrolize the glycans. While samples are incubating, again, prepare the evaporation manifold at 75 degrees celsius.After incubation, dry the samples under a steady stream of nitrogen in the evaporation manifold. Frequently check samples to ensure they are dried using the minimum amount of drying time possible. To each sample, add 475 micro-liters of 10 grams per liter sodium borohydride.And then mix samples thoroughly to dissolve any residue. Cap the test tubes and allow the reduction reaction to continue for one hour. Next, add 63 micro-liters of methanol to each sample.Mix and then dry the samples at 75 degrees celsius under nitrogen. Add 125 micro-liters of methanol to acetic acid solution to each sample. Mix and then dry the samples again at 75 degrees celsius under nitrogen.Ensure samples are completely dry by placing under a vacuum for 20 minutes. After drying, add 18 micro-liters of deionized water to each sample. Then centrifuge the samples briefly and mix them well to dissolve an precipitate.Next, add 250 micro-liters of acetic anhydride to each tube. Cap the tubes, thoroughly vortex and sonicate the samples in a water bath for two minutes to ensure complete dissolution of any residue. Then incubate the samples at 50 degrees celsius for 10 minutes.Now add 230 micro-liters of concentrated TFA to each sample. After capping and mixing the samples, incubate them for 10 minutes an internal temperature of 50 degrees celsius. Add 1.8 milliliters of dichloromethane to each sample and mix well.Next, add two milliliters of dionized water to the tube and perform liquid/liquid extraction twice. Recover the dichloromethane layer with a silanized pasture pipe head. Transfer to a auto-sampler vial and then dry the sample under nitrogen at 74 degrees celsius.Prior to GC-MS, reconstitute the samples each with 120 micro-liters of acetone, capping and mixing them thoroughly. In split mode, use an auto-sampler to inject one micro-liter of each sample into the GC split-mode liner. For time of flight mass spectrometers, a split ration of forty and helium gas in constant flow mode at 0.8 milliliters per minute is recommended.Analyze the sample using a two-step gradient that reaches 325 degrees celsius. Subject the eluding sample components to electron ionization. For a time of flight mass analyzer, analyze fragments M over Z 40 to 800 with a 0.1 second pulse summation rate.To demonstrate the sensitivity of this protocol, a biological sample was processed carefully. The resulting chromatogram gives adequate intensity between the desired product peaks for ease of comparison and analysis. The same starting biological sample was then processed with the same procedure.But the white residue in the permethylation solution that was spun through the NAOH column was carried into the subsequent mixture for liquid/liquid extraction. The chromatogram gives a different ratio of peak intensity of the same key sample products. When compared directly, the difference is stark.The clear depression of the 4-position linked N-acetylglucosamine peak in second chromatogram is a recognizable trait of an unsuccessful permethylation and extraction. To appreciate the utility of this approach toward detecting differences in glycan composition in disease states, extracted ion chromatograms from a normal blood plasma sample and a cancer patient blood plasma sample, are normalized for 2-linked manos and overlaid. Quantitative differences in unique glycan features such as beta 1-6 branching as well as sick cyalalation are readily apparent.After watching this video, you should have a good understanding of how to carry out bottom up glycomics glycan node anaylsis. Facilitating distillation of interesting glycan features such as corfulcosulation, sick cyalalation and bisecting glycanite into single analytical signals.

glycan-node-analysis-a-bottom-up-approach-to-glycomics

Research

JoVE Journal

Chemistry

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.

A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening.

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

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 ...

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Original Experimental Approach for Assessing Transport Fuel Stability

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several technological changes to fulfill environmental and economic requirements. These developments have resulted in increasingly severe operating conditions whose suitability for conventional and alternative fuels needs to be addressed. For example, fatty acid methyl esters (FAMEs) introduced as biodiesel are more prone to oxidation and may lead to deposit formation. Although several methods exist to evaluate fuel stability (induction period, peroxides, acids, and insolubles), no technique allows one to monitor the real-time oxidation mechanism and to measure the formation of oxidation intermediates that may lead to deposit formation. In this article, we developed an advanced oxidation procedure (AOP) based on two existing reactors. This procedure allows the simulation of different oxidation conditions and the monitoring of the oxidation progress by the means of macroscopic parameters, such as total acid number (TAN) and advanced analytical methods like gas chromatography coupled to mass spectrometry (GC-MS) and Fourier Transform Infrared - Attenuated Total Reflection (FTIR-ATR). We successfully applied AOP to gain an in-depth understanding of the oxidation kinetics of a model molecule (methyl oleate) and commercial diesel and biodiesel fuels. These developments represent a key strategy for fuel quality monitoring during logistics and on-board utilization.

Oxidation stability of transport fuels has become a concern for future fuel development. This work presents an original methodology developed by IFP Energies Nouvelles for assessing fuel stability using two different reactors. This methodology was successfully applied to gain an in-depth understanding of the oxidation kinetics and pathways of model molecules and commercial fuels.

The study of fuel oxidation stability is an important issue for the development of future fuels. Diesel and kerosene fuel systems have undergone several ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Synthesis of pH Dependent Pyrazole, Imidazole, and Isoindolone Dipyrrinone Fluorophores using a Claisen-Schmidt Condensation Approach

Methine-bridged conjugated bicyclic aromatic compounds are common constituents of a range of biologically relevant molecules such as porphyrins, dipyrrinones, and pharmaceuticals. Additionally, restricted rotation of these systems often results in highly to moderately fluorescent systems as observed in 3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidin-3-ones, xanthoglows, pyrroloindolizinedione analogs, BODIPY analogs, and the phenolic and imidazolinone ring systems of Green Fluorescent Protein (GFP). This manuscript describes an inexpensive and operationally simple method of performing a Claisen-Schmidt condensation to generate a series of fluorescent pH dependent pyrazole/imidazole/isoindolone dipyrrinone analogs. While the methodology illustrates the synthesis of dipyrrinone analogs, it can be translated to produce a wide range of conjugated bicyclic aromatic compounds. The Claisen-Schmidt condensation reaction utilized in this method is limited in scope to nucleophiles and electrophiles that are enolizable under basic conditions (nucleophile component) and non-enolizable aldehydes (electrophile component). Additionally, both the nucleophilic and electrophilic reactants must contain functional groups that will not inadvertently react with hydroxide. Despite these limitations, this methodology offers access to completely novel systems that can be employed as biological or molecular probes.

The Claisen-Schmidt condensation reaction is an important methodology for the generation of methine-bridged conjugated bicyclic aromatic compounds. Through utilizing a base-mediated variant of the aldol reaction, a range of fluorescent and/or biologically relevant molecules can be accessed through a generally inexpensive and operationally simple synthetic approach.