• Preparations
  • Modification
  • Profile
  • Composition
  • Miscellaneous
Full catalog pdf of catalog

Pre-001: Preparation of mixed N-Glycans.

The starting material for this procedure can be purified protein, cell extract or tissue. Protein is solubilized and denatured with SDS/2-ME. NP-40 is added to "buffer" the SDS. PNGase F is added and the mixture incubated overnight at 37°C. The mixture is applied to a Sep-Pak C18 cartridge equilibrated in water. Proteins and the majority of detergent bind to the resin. The run through and water washes are applied to a porous graphitized carbon (PGC) cartridge. Under aqueous conditions, oligosaccharides bind to the PGC cartridge, while salts and buffers pass through unretarded. Carbohydrates are eluted with 30% acetonitrile, 0.1% TFA in water while any residual detergent remains bound to the cartridge. The resulting desalted, N-glycans are dried and ready for further analysis.

Pre-002: Preparation of Mixed O-Glycans

The starting material for this procedure can be purified protein, cell extract or tissue. The sample is incubated with 0.1 M NaOH/1M NaBH4 at 45°C overnight to release O-glycans by way of alkaline induced β-elimination. The sample is passed through a cartridge packed with Dowex 50 (H+ form) to remove peptide and Na+. The flow through is collected and lyophilized. Repeated addition of methanol/acetic acid to form volatile methylborates followed by drying under a stream of nitrogen yields desalted, dried, reduced O-glycans ready for further analysis.

Pre-003: Preparation of Mixed Glycolipid-derived Glycans

The starting material for this procedure can either be purified glycolipids or crude lipid extracts, e.g., Pre-004. Lipid mixtures are treated with endoglycoceramidase to release the glycan from the lipid. The mixture is passed over a Sep-Pak C18 cartridge to remove enzyme and lipid and the run-through is applied to a PGC cartridge. Under aqueous conditions, oligosaccharides bind to the PGC cartridge, while salts and buffers pass through unretarded. Carbohydrates are eluted with 30% acetonitrile, 0.1% TFA in water while any residual detergent remains bound to the cartridge. The resulting desalted, glycolipid-derived glycans are dried and ready for further analysis.

Pre-004: Preparation of Total Lipid Extracts

The starting material for this procedure is typically tissues. Tissues are sequentially extracted with chloroform: methanol, twice with each of the following ratios 2:1, 1:1 and 1:2. The pooled extracts are dried and resuspended in chloroform: methanol 2:1.

Pre-005: Preparation of Cellular Metabolites-TCA Extraction

The starting material for this procedure is typically tissue or cell pellets. Freshly isolated material is preferable. Samples are treated with TCA (5-10% w/v final). The precipitated macromolecules are removed by centrifugation. The supernatant is treated with an equal volume of Freon: trictylamine (3:1) to extract the TCA and the aqueous phase is stored frozen or lyophilized.

Pre-006: Preparation of Glycosaminoglycans

The starting material for this procedure is typically tissue or cell pellets. Hard tissues are grinded on liquid nitrogen or soft tissues are homogenized on ice bath on protease treatment buffer. Samples are digested overnight with Pronase/ Protease at 37°C and centrifuged at 14000rpm for 20min. The supernatant is passed through a DEAE column and the bound glycosamino glycan (GAG) is eluted with 2M NaCl. The GAG is then desalted on PD10 size exclusion column and purified GAG is lyophilized and used for further analysis.

Pre-007: Preparation of Glycopeptides

Protein samples are treated with Trypsin, at 37°C, 16-18h, followed by destruction of the enzyme by immersing the sample in 100°C for 5min. The sample is then passed through Sep-Pac C18 column and the glycoprotein fraction is eluted at 15-30% Acetonitrile fraction.

Pre-008: Analysis of Sulfate and Phosphate

Oligosaccharides and glycoconjugates may contain covalently attached non-carbohydrate groups; among those, sulfate and phosphate are quite common. The presence and quantity of those groups is determined by subjecting the sample to pyrolysis, followed by autosampler injection and separation of the anions on an ion-exchange (Dionex IonPac) column and detected by Suppressed conductivity detector. Samples are hydrolyzed using 0.3N HCl at 150°C for 30min. Hydrolyzed samples are dried on speed vac and pyrolsed for 15Sec, cooled dissolved in water and injected on the Dionex IonPac column.

Pre-009: Extraction of Semi-Rough or Smooth LPS (Hot Phenol water extraction)

The bacterial cells was washed by PBS once and then twice with distilled water to remove the adhered media. The cells were suspended in water (7mL water per gm or bacterial cell) and sonicated for 1 min to make the cell-suspension. The cell suspension was stirred on oil/or water bath maintained at 65°C for 10min using a magnetic stirrer. 7mL of 90% phenol was pre-heat to 65°C and added to the aqueous bacterial suspension. The mixture was stirred for 30min at 65°C and immediately cooled down below 10°C on an ice-water bath. The reaction mixture was centrifuged at 4000rpm for 40min at 10°C. The top layer containing Smooth LPS was removed carefully and dialyzed against distilled water for 3-4days with changing of water each day until the smell of phenol was gone. The extracted LPS were treated with DNase/RNase and Proteinase K to remove contaminating nucleic acid and proteins respectively. Finally the LPS were precipitated by ultracentrifugation at 120,000g for 6h at 5°C.

Pre-010: Extraction of Rough LPS (Phenol-Chloroform-Petroleum Ether extraction method)

Bacterial calls was washed with deionized water (once), 90% ethanol (twice), acetone (twice) and di-ethyl ether (once) and dried in vacuum desiccators. The dried cells were grinded to fine powder and reaction mixture in the ratio of 2:5:8 of 90% phenol: chloroform: petroleum-ether (40-60°C fractions) was added (200mL of reaction mixture for 50gm of dried cell or equivalent). This cell-solvent mixture was stirred at high speed maintaining the temperature near 10°C for 30min. The extracted material was centrifuged at 3500rpm for 10min at 10°C. Supernatant was collected and chloroform and petroleum ether was removed from the mixture by rotary evaporation. Rough LPS was precipitated from the phenol layer by careful addition was water dropwise. Precipitated LPS was centrifuged and repeatedly washed with 5:1 Di-ethyl ether: acetone mixture and dried by Nitrogen flush.

Pre-011: Capsular polysaccharide extraction

Cells were shaken with PBS or 1% aqueous phenol for 3h at room temperature and centrifuged at 7000RPM for 20 min at 5°C. Crude CPS was isolated from supernatant by cold absolute ethanol precipitation. CPS was removed by centrifugation at 14000RPM for 20min at 5°C. The precipitate was treated with Nuclease and Proteinase K and dialyzed against water. Enzyme treated CPS was further purified by size exclusion chromatography or weak anion exchange chromatography.

Pre-012: Bacterial Lipid Extraction

Bacterial lipids are extracted from peletted cells using Bligh-Dyers extraction protocol. Pellet cells approximately 1x107 cells are suspended in 3mL CHCl3: CH3OH (3:1) mixture and vortexed thoroughly. 0.8mL of water was added again vortexed and allow the solution tow sit at room temp for at least 30min. Pellet cellular debris by spinning at 3000 rpm for 5min. Transfer the solvents to a new tube without transferring any of the pelleted debris. Add 1mL of CHCl3 and 1mL water to each sample (upon addition of CHCl3 a biphasic solution should result). Vortex hard and spin samples at 3000 rpm for 5min. Remove the upper aqueuous/ methanolic phase and transfer the lower chloroform layer to a glass tube and dry down using dry nitrogen flush. The lipid residues are stored at -80°C for future analysis.

Pre-013: Sugar nucleotide extraction

Cells were pelleted by centrifugation washed once in ice cold phosphate buffered saline and then lysed by sonication or lysis buffer. The lysed cells were centrifuged at 16000 rpm for 10 min at 4°C to remove insoluble material and the supernatant was dried under nitrogen. The samles were dissolved in 200ul of 9% butanol and extracated three times with 400ul of 90% butan-1-ol to remove lipids. The resulting aqueous phase was dried under nitrogen and resuspended in 5mM ammonium bicarbonate and sugar nucleotides were extracted using EnviCarb graphitized carbon columns (Supelco). Columns were prepared by washing with 3mL of 80% acetonitrile, 0.1% triflouroacetic acid followed by 2mL water. The samples was loaded in 5mM ammonium bicarbonate and the column was washed with 2mL water, 2mL 25% acetonitrile and 2mL 50mM TEAA buffer and finally eluted with 2mL of 25% acetonitrile, 50mM TEAA buffer. Eluate was freeze dried and stored at -80°C prior to analysis.
Full catalog pdf of catalog

Mod 001: Alditol Acetate derivative for GC-MS (AA)

Dry glycan samples were hydrolysed by 4N TFA at 100°C for 4h, followed by removal of the acid by dry nitrogen flush. The acid was removed complete by coevaporation twice using 1:1 isopropanol: water mixture under dry nitrogen flush. Hydrolysed samples are reduced overnight by Sodium borohydride in 1M ammonium hydroxide solution. Excess borohydride was neutralized by 30% acetic acid and boric acid was removed as their methyl borate. Samples are finally treated with 1:1 acetic anhydride: pyridine mixture at 100°C for 1h. Pyridine and acetic anhydride was removed by nitrogen flush and alditol acetate was extracted with dichloromethane.

Mod 002: Trimethyl Silyl derivative for GC-MS (TMS)

The starting material for this procdure is typically glycoprotein, glycolipid or glycan. Buffer and salt should be minimized. Samples should be completely dried or lyophilized. For 0.1-0.25mg of sample 250μl 1M Methanolic Hydrochloric was added and methanolysed at 80°C for 16-18h. Methanolic hydrochloric acid was removed by dry nitrogen flush, and coevaporated one more time with dry methanol. Samples are re-N-acetylated by 4:1:1 methanol: pyridine: acetic anhydride at 100°C for 1h. Reagents were removed by dry nitrogen flush and finally treated with Tri-Sil (Pierce) reagent at 80°C for 30min. The TMS derivative of the monosaccharides was extracted with hexane and ready for injection on GC-MS.

Mod 003a: Per-O-Methylation of glycans (PM)

The starting material for this procedure should be purified and completely dried glycolipids or glycan. Samples are dissolved in dry DMSO and stirred for several hours until the sample is completely dissolved. Powdered Sodium hydroxide or Sodium hydroxide slurry in DMSO was added as base and kept for vigorous stirring for 1-2h. Samples were cooled on an ice bath and 200μl of methyl iodide was added and continued stirring for 1h. Another aliquote of 100μl of methyl iodide was added and kept stirring for 30min. To the reaction mixture 0.5mL of chloroform was added and stirred for 10min and the reaction was stopped by adding 1mL of water. The methylated glycan was extracted in the chloroform layer, dried and used for further analysis by MALDI mass spectrometry or as PMAA derivative for linkage analysis.

Mod 003b: Partially methylated Alditol Acetate (PMAA)

Partially methylated alditol acetate derivative is done after permethylation of glycans as described in Mod 003a. The PM samples are hydrolyzed by 4N TFA at 100°C for 6hours. Followed by removal of the acid, reduction and acetylation as described in Mod-001.

Mod 004: Fatty acid methyl ester (FAME)

Dried samples are methanolysed by 1M methanolic HCl at 80°C for 18h. The acid was removed by nitrogen flush and half saturated NaCl was added to the samples and fatty acid methyl ester was extracted using chloroform. The chloroform layer was back extracted with water and was dried by nitrogen flush. Fatty acid samples were dissolved in hexane and injected on GC-MS.

Mod 005: Mild acid treatment for releasing Sialic acid

The starting material for this procedure is typically isolated glycan, glycolipids, although glycopeptides and intact proteins may also be deemed appropriate depending on the nature of the sample. Sample is dissolved in a final concentration of 2 M HOAc and heated to 80°C for 3 hours. Acetic acid is removed by drying.

Mod 006: De-O-Acetylation of glycans

De-O-Acetylation of glycans are achieved by treating the samples with base 50mM of Sodium hydroxide at room temp for 3h or by treatment with anhydrous hydrazine. Sodium hydroxide was neutralized by 30% acetic aicd under cold condition and the glycan was purified by PD10 size exclusion chromatography. Anhydrous hydrazine treated material was precipitated by cold (-80°C) acetone the precipitate was washed once more time with cold acetone and finally the precipitate was dissolved in water and lyophilized.

Mod-007: Enzymatic degradation of Chondroitin Sulfate (GAG-CS)

The starting material for this procedure is dried, desalted glycosaminoglycan. Glycosaminoglycans are dissolved in buffer appropriate for the lyase(s) to be used. A cocktail mixture of Chondroitinase ABC is added and the polysaccharide is digested at 37°C for 18hours. Following digestion, the mixture is fractionated using a 10,000 MWCO filter to remove enzyme and undigested glycosamino-glycan chains. The supernatant is dried and used for Pro-004 or Mod-009

Mod-008: Enzymatic degradation of Heparin Sulfate (GAG-HS)

The starting material for this procedure is dried, desalted glycosaminoglycan. Glycosaminoglycans are dissolved in buffer appropriate for the lyase(s) to be used. A mixture of Heparinase I, II and III is added and the polysaccharide is digested at 37°C for 18hours. Following digestion, the mixture is fractionated using a 10,000 MWCO filter to remove enzyme and undigested glycosamino-glycan chains. The supernatant is dried and used for Pro-005 or Mod-009.

Mod-009: Isotopic Aniline tagging for GAG disaccharide analysis by mass spectrometry (GRIL-Glycan Reductive Isotope Labeling)

1 pmol to 10 nmol of HS and CS disaccharides was transferred to 1.5-ml Microcentrifuge tubes and dried down in a centrifugal evaporator. 15 µl of 12C6 aniline or 13C6 aniline and 15 µl of 1 M NaCNBH3 (Sigma-Aldrich) freshly prepared in dimethyl sulfoxide: acetic acid (7:3, v/v) were added to each sample. Reactions were carried out at 65°C for 4h or alternatively at 37°C for 16 h and then dried in a centrifugal evaporator. The dried samples are resuspended in running buffer and used for LCQ-MS analysis (Pro-009).

Mod-010: 2AB labeling of glycans

For labeling with 2-Aminobenzamide (2-AB) 100 ul of the cyanoborohydride mixture (prepared by adding 6.5 mg of sodium cyanoborohydride in 200µl of 35:65 glacial acetic acid: DMSO mixture) was added to 6 mg of 2-AB reagent, vortexed and sonicated to mix thoroughly. 10 ul of 2AB reagent was added to each sample and incubated at 65°C for 2.5 h. After 2AB labeling the samples were purified by passing through a Glycoclean S-cartridge dried on a speed vac and was ready for further analysis.
Full catalog pdf of catalog

Pro-001: HPAEC-PAD Profile of N-Glycans

The starting material for this procedure is a mixture of desalted N-glycans, e.g., Pre-001. Reduced N-glycans can be similarly analyzed. The sample is dissolved in water and profiled by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) using either a Dionex Carbo-Pac PA-1 or PA-100 column. Increasing concentrations of NaOH and NAOAc effect elution. Species elute in order of increasing negative charge. Elution time is also affected by the size of the oligosaccharide, the nature of the glycosidic linkages and the presence of fucose residues. Control analysis consists of profiling a standard mixture of fetuin for sialylated N-glycans and RNaseB for high mannose N-glycans.
Example

Pro-002: O-Glycan profiling

The starting material for this procedure is a mixture of desalted (generally reduced) O-glycans, e.g., Pre-002. The sample is dissolved in water and profiled by High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) using either a Dionex Carbo-Pak PA-1 or PA-100 column. Increasing concentrations of NaOH and NaOAc effect elution. Species elute in order of increasing negative charge. Elution time is also affected by the size of the oligosaccharide, the nature of the glycosidic linkages and the presence of fucose residues. Gradient conditions differ from those used for N-glycans. Control analysis consists of profiling a standard mixture of either bovine submaxillary gland O-glycans, or milk oligosaccharides.
Example

Pro-003: 2AB-labelled glycan profiling

2-AB labeled glycans are separated by HPAEC and detected by fluorescence detection; this is a very sensitive method and can determine the glycans in pico-molar levels. 2-AB labeled glycans are dissolved in water and separated on PA-1 or PA-100 column using sodium hydroxide and sodium acetate gradient. The detection is done by an online fluorescence detector.
Example

Pro-004: Profile of Glycosaminoglycan-derived Disaccharides by Ion Pair RP-HPLC with UV and Fluorescent Detection

The starting material for this procedure is typically a mixture of disaccharides obtaining by the action of lyases. This method is prefered for chondroitin- and dermatan-derived disaccharides. Disaccharides are adsorbed to the C18 column by virtue of their ionic interactions with the hydrophobic ion pair reagent tetrabutylammonium hydrogen sulfate. Elution is effected with a gradient of increasing acetonitrile concentration. Disaccharides are detected by the absorbance at 232 nm due to the presence of the double bond resulting from the action of the lyases. UV detection has a detection limit of ~100 pMoles. The effluent from the UV detector is mixed with NaOH and 2-cyanoacetamide, heated to 130°C to form a fluorescent adducts and then detected fluorometrically. The limit of detection of the fluorscence detection is ~5 pMoles. Control analysis consists of a mixture of known glycosaminoglycan-derived disaccharides.
Example

Pro-005: Profile of Glycosaminoglycan-derived Disaccharides by Anion Exchange HPLC with UV and Fluorescent Detection

The starting material for this procedure is typically a mixture of disaccharides obtaining by the action of lyases. This method is prefered for heparin- and heparan sulfate-derived disaccharides. Elution is effected with a gradient of increasing NaCl concentration. Disaccharides are detected by the absorbance at 232 nm due to the presence of the double bond resulting from the action of the lyases. UV detection has a detection limit of ~100 pMoles. The effluent from the UV detector is mixed with NaOH and 2-cyanoacetamide, heated to 130°C to form a fluorescent adducts and then detected fluorometrically. The limit of detection of the fluorscence detection is ~5 pMoles. Control analysis consists of a mixture of known glycosaminoglycan-derived disaccharides.
Example

Pro-006: Single HPLC run

(No sample preparation based on hourly charge)
Example

Pro-007: MALDI-TOF profiling of glycans

Starting material would be purified glycans, permethylated glycans or glycolipids. Native glycans are dissolved in water at a concentration of 2µg/µl and mixed in 1:1 ratio with DHB as matrix and spotted on the MALDI plate. Permethylated glycans are dissolved in absolute methanol and mixed with matrix in 1:1 ratio and spotted on the MALDI plate. Glycolipid samples are dissolved in 3:1 Chloroform: Methanol mixture before spotting. The samples are air dried and analyzed in positive mode, in order to do a negative mode analysis for samples which bear sialic acids or negative ions such as sulfate and phosphate THAP was used as matrix.
Example

Pro-008: Cellular metabolite analysis

The starting material for this procedure is typically a TCA extract from cells or tissues, e.g. Pre-005. The mixture is profiled by anion exchange chromatography using a Dionex PA-1 column equilibrated in either water or 10mM NaOH. Elution is effected by a gradient of increasing NaOAc concentration. UV detection is typically performed at 260 nm. The dominant species present in this analysis are typically nucleotide phosphates. Sugar nucleotides are well resolved using a combination of the two gradient systems described above, however true estimates of sugar nucleotide concentrations may first require the removal of nucleotide phosphates. Detection limits of ~1 nMoles/peak are typical. Control analysis consists of profiling a standard mixture of sugar nucleotides.
Example

Pro-009: GRIL LCQ-MS for GAG disaccharide Analysis

An LCQ classic quadrupole ion trap mass spectrometer equipped with an electrospray ionization source, and a quaternary high-performance liquid chromatography pump (Thermo-Finnigan, San Jose, CA) is used for disaccharide analyses. Aniline isotopic and non-isotopic disaccharides prepared by Mod-009 are separated on a C18 reversed-phase column (0.46 cm x 25 cm, Vydac) with the ion pairing agent dibutylamine (DBA, Sigma-Aldrich). The solvent gradient used for eluting the samples are: 100% buffer A (8mM acetic acid, 5mM DBA) for 10 min, 17% buffer B (70% methanol, 8 mM acetic acid, 5 mM DBA) for 15 min; 32% buffer B for 15 min, 40% buffer B for 15 min, 60% buffer B for 15 min; 100% buffer B for 10 min; and 100% buffer A for 10 min. The most highly substituted disaccharides elute at 60% buffer B (42% methanol). Ions of interest are monitored in negative ion mode, and signal intensity is optimized for a representative species of disaccharide. To minimize in-source fragmentation of sulfated disaccharides, the capillary temperature and spray voltage were kept at 140 °C and 4.75 kV, respectively.
Full catalog pdf of catalog

Com-001: Monosaccharide analysis by HPAEC-PAD

The starting material for this procedure is typically glycoprotein, glycolipid or glycan. Buffer and salt should be minimized. Samples (0.1mg) are treated with 200µl of 2 M TFA at 100°C for 4h to cleave all glycosidic linkages. Acid is evaporated by dry nitrogen flush, repeated co-evaporation with 1:1 isopropanol: water helps to get rid of the acids completely. Glycolipid and glycoprotein samples can be passed through SepPak C18 cartridge and evaporated to remove the acid. Finally the samples are dissolved in water and analyzed by HPAEC-PAD using a CarboPac PA-1, PA-10 or PA-20 column. Monosaccharide standards found are treated in parallel and used for calibration of HPAEC-PAD response.
Example

Com-002: Uronic acid analysis by HPAEC-PAD

The starting material for this procedure is typically any proteoglycan, plant polysaccharide or bacteria derived glycans. Dried samples are treated with 2M TFA at 100°C for 6 h to cleave all glycosidic linkages. Alternatively, methanolysis may be used to hydrolyze glycosidic bonds and concomitantly form the methyl glycoside and methyl ester derivatives. Methanolysis is followed by 0.5N TFA hydrolysis to remove these methyl groups at 100°C for 1h. After drying and removing the acids from the hydrolyzate, samples are dissolved in water and analyzed by HPAEC-PAD using a CarboPac PA-1 column. Common uronic acid standards (Glucuronic, Galacturonic and iduronic acids) are treated in parallel and used for calibration of HPAEC-PAD response.
Example

Com-003: Sialic acid analysis by HPAEC-PAD

The starting material for this procedure can be glycoprotein, glycolipid or glycan. Sample is dissolved in a final concentration of 2 M HOAc and heated to 80°C for 3 hours to release sialic acids. The released sialic acids are collected by ultra-filtration through a 3,000 MWCO filter, dried and analyzed by HPAEC-PAD using a Dionex CarboPac PA-1 column eluted with a sodium acetate gradient that separates N-acetylneuraminic acid and N-glycolylneuraminic acid. As little as 100 picomol of individual sialic acid can be detected. Known standards are run in parallel, sialic acids are identified by elution position and quantitation is done in reference to known amounts of N-acetylneuraminic acid injected in parallel. The printout of the results shows the profile and individual sialic acid content expressed in nanomoles present in the volume injected. Blanks (that represent background from the methodology used for sample preparation) are required for optimum quantitative results.
Example

Com-004: Analysis of Anions by Ion Chromatography

The starting material for this procedure can be glycoprotein, glycolipid or glycan. Oligosaccharides and glycoconjugates may contain covalently attached non-carbohydrate groups; among those, sulfate and phosphate are quite common. The presence and quantity of those groups is determined by subjecting the sample to pyrolysis, followed by autosampler injection and separation of the anions on an ion-exchange (Dionex IonPac) column. Conductivity detector was used in conjunction with a suppressor to get optimum results. Blanks (that represent background from the methodology used for sample preparation) are required and it is recommended that samples be run in duplicate for optimum quantitative results.
Example

Com-005: Reverse phase HPLC of DMB-Sialic acid and determination by Fluorescence detector

The starting material for this procedure can be glycoprotein, glycolipid or glycan. Sample is dissolved in a final concentration of 2 M HOAc and heated to 80°C for 3 hours to release sialic acids. The released sialic acids are collected by ultra-filtration through a 3,000 MWCO filter and derivatized with DMB. The fluorescent sialic acid derivatives are analyzed by reverse-phase HPLC with on-line fluorescence detection. The method can detect as low as 250 femtomol of individual sialic acid. Identification is based on known standards run in parallel, and quantitation is done in reference to known amounts of N-acetylneuraminic acid derivatized and injected in parallel.
Example

Com-006: GC-MS of TMS derivative

Trimethylsilyl derivatives of methyl glycosides of neutral and N-Acetylated amino sugars and trimethylsilyl derivative of methyl glycoside methyl ester of uronic acids are successfully separated by GC-MS using either DB-1 or DB-5 capillary column. TMS sugars are detected from their retention time and by electron impact (EI) ionization mass spectrum. Analysis requires standards to be prepared and analyzed in parallel. The aforementioned monosaccharides are identified by elution position and fragmentation profile. Blanks (that represent background from the methodology used for sample preparation) are required for optimum quantitative results.
Example

Com-007: GC-MS of AA derivative

Alditol acetate derivative of neutral and amino sugars are separated on DB-1 capillary column. Neutral pentoses and deoxy-hexose sugars are better resolved in SP-2330 capillary column. AA sugars are detected from their retention time and by electron impact (EI) ionization mass spectrum. Analysis requires standards to be prepared and analyzed in parallel.
Example

Com-008: GC-MS of PMAA derivative (linkage analysis)

The starting material for this procedure should be purified glycolipid or glycan. These are methylated according to the Ciucanu-Kerek or Hakomori methods as appropriate. Purification methods used include extraction, Sep-Pak cartridge purification, dialysis or chromatography over LH-20 depending on the sample. Permethylated products are cleaved by acetolysis/hydrolysis (when amino sugars are present) or acid hydrolysis. Partially methylated monosaccharides are reduced, and partially methylated alditols are derivatized to obtain partially methylated alditol acetates. Analysis of these products is done by GC-MS using DB-5 or equivalent capillary column. Identification was achieved by using a combination of retention times (as compared to those of known standards analyzed under the same conditions) and mass fragmentation pattern.
Example

Com-009: GC-MS of Fatty acids

Fatty acid methyl esters are dissolved in hexane and analyzed by GC-MS in EI or CI mode. DB-1 or DB-5 can separate the fatty acids and the mass fragmentation patterns are used to identify saturated and unsaturated fatty acids. Hydroxy-fatty acids are components of bacterial lipids and can be detected by characteristic mass fragmentation pattern. The hydroxyl fatty acids are treated with Tri-Sil there by forming trisilyl methyl ether of fatty acid methyl ester.
Example

Com-010: LCQ-MS Single runs no sample prep (Per hour)

Suitable sample are dissolved in 1:1 methanol: water mixture containing 0.1% formic acid and data collected on positive mode. For negative mode ammonium hydroxide is added on the solvent mixture. The liquid chromatography was done using suitable column and solvent buffer compatible with mass spectroscopy.

Com-011: Glycan analysis by LCQ-MS

Glycan samples are separated on suitable column (reverse phase or amino- bonded column) connected online to the mass spectroscope. Acetonitrile: water mixture containg ammonium fromate or formic acid can be used as mobile phase for amino- bonded column and Methanol: acetonitrile and water was used for reverse phase columns.

Com-012: Nucleotide sugar analysis by HPAEC-UV

The mixture of sugar nucleotide was profiled by anion exchange chromatography using a Dionex PA-1 column equilibrated in either water or 10mM NaOH. Elution is effected by an increasing gradient of NaOAc concentration. UV detection is typically performed at 260 nm. The dominant species present in this analysis are typically nucleotide phosphates. Sugar nucleotides are well resolved using a combination of the two gradient systems described above, however true estimates of sugar nucleotide concentrations may first require the removal of nucleotide phosphates. Detection limits of ~1 nMoles/peak are typical. Control analysis consists of profiling a standard mixture of sugar nucleotides.
Example
Full catalog pdf of catalog

Mis-001: Consultation Time (Per Hour)

It is the policy of the Glycotechnology that each project be given a maximum of 30 minutes of discussion at no charge with the Core Director. Additional time for individualized analysis of the problem, help in selecting the best protocol for the isolation, purification and analysis of glycoconjugates and interpretation of data is available through consultation with the Core Director, depending upon available time.

Mis-002: Additional Analytical Time (Per Hour)

There may be an additional charge if custom methods are requested or if there is any analysis which is not listed on the website. This charge will not be imposed without prior consultation with the investigator.

Mis-003: Method development based on time and materials used

Glycotechnology core is always willing to do a method development and find out new strategies for analyzing glycans. This can be done upon request, however the customer have to provide with materials or columns if needed. There would always be a charge for establishing the new method and handing it over to the customers.

Mis-004: Fraction collection by HPLC (Per Fraction)

Glycotechnology core can collect fractions from HPLC for the customers for further analysis or send them to customers. The customers have to pay for the shipping charges.

Mis-005: Powerpoint slide preparation.

Many of the instruments in the Glycotechnology Core utilize proprietary software that is not commonly found in most research laboratories. We therefore can provide any data profile in Microsoft PowerPoint thus eliminating the need to scan data printouts.

Mis-006: NMR analysis on the glycan samples will be done upon request.

Contact the Core Director for a price quote.
Glycotechnology Core Resource • University of California, San Diego • 9500 Gilman Drive, BRF2, Room 4243, La Jolla, CA 92093-0687