Description Continued

Composition Analysis Com-001. Composition Analysis of Neutral Amino Sugars by HPAEC-PAD.

The starting material for this procedure is typically glycoprotein, glycolipid of glycan. Buffer and salt should be minimized. Glycoproteins in solution glycolipids are treated with 2 M TFA at 100°C for 4 h to cleave all glycosidic linkages. After drying the hydrolyzate, samples are dissolved in water and analyzed by HPAEC-PAD using a CarboPac PA-1 column eluted with a low concentration of sodium hydroxide. Common monosaccharide standards found in vertebrate oligosaccharides (Man, Gal, Glc, GlcNAc, GalNAc, Fuc and Xyl) are treated in parallel and used for calibration of HPAEC-PAD response. The printout of the results shows the profile and individual monosaccharide content expressed in nanomoles present in the volume injected. The aforementioned monosaccharides are identified by elution position. Blanks (that represent background from the methodology used for sample preparation) are required for optimum quantitative results.

Com-002. Composition Analysis of Sugar Alcohols (Alditols) by HPAEC-PAD.

The starting material for this procedure is typically glycoprotein, glycolipid of glycan. Buffer and salt should be minimized. Glycoproteins in solution glycolipids are treated with 2 M TFA at 100°C for 4 h to cleave all glycosidic linkages. After drying the hydrolyzate, samples are re-N-acetylated in sodium bicarbonate using acetic anhydride. The mixture is then reduced and desalted as described above for Mod-001. Alternatively, O-GlcNAcitol, O-GalNAcitol and O-Fucitol can be quantified following alkaline induced -elimination as described above in Pre-002. Desalted sugar alcohols (alditols) are dissolved in water and directly injected in a CarboPac MA-1 column, eluted with a gradient of sodium hydroxide and monitored by PAD. The method separates amino sugar alcohols (both in their N-acetylated and free amino forms), fucitol, xylitol, glucitol, and mannitol from each other and from the corresponding reducing monosaccharides. Common alditol standards found in vertebrate oligosaccharides (Mannitol, Galactitol, Glcitol, GlcNAcitol, GalNAcitol, Fucitol and Xylitol) are treated in parallel and used for calibration of HPAEC-PAD response. The printout of the results shows the profile and individual alditol content expressed in nanomoles present in the volume injected. The aforementioned alditols are identified by elution position. Blanks (that represent background from the methodology used for sample preparation) are required for optimum quantitative results.

Com-003. Composition Analysis of Neutral, Amino Sugars as TMS derivatives by GC-MS.

The starting material for this procdure is typically glycoprotein, glycolipid of glycan. Buffer and salt should be minimized. This method is preferred to Com-001 when there is a large amount of peptide relative to carbohydrate, when unusual monosaccharides are expected or when mass spectrometric confirmation of common monosaccharides is required. Methanolic HCl is used to hydrolyze glycosidic bonds and concomitantly form the methyl glycoside derivative (methanolysis). Free hydroxyl groups are trimethylsilylated using Tri-SilTM and the resulting volatile derivatives are fractionated by gas chromatography on a DB-1 column using a temperature gradient and detected by electron ionization mass spectrometry. Analysis requires standards to be prepared and analyzed in parallel. he printout of the results shows the profile and individual monosaccharide content expressed in nanomoles present in the volume injected. 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.

Com-004. Uronic Acid Determination: HPAEC-PAD.

The starting material for this procedure is typically any glycoprotein, glycolipid of glycan suspected of having a uronic acid present. Buffer and salt should be minimized. Glycoproteins in solution glycolipids are treated with 2 M TFA at 100°C for 6 h to cleave all glycosidic linkages. Alterantively, methanolysis may be used to hydrolyze glycosidic bonds and concomitantly form the methyl glycoside and methyl ester derivatives. Methanolysis is followed by TFA hydrolysis to remove these methyl groups. After drying 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. The printout of the results shows the profile and individual uronic acid content expressed in nanomoles present in the volume injected. The aforementioned uronic acids are identified by elution position. Blanks (that represent background from the methodology used for sample preparation) are required for optimum quantitative results.

Com-005. Sialic Acid Determination as DMB Derivatives by RP-HPLC with Fluorescence Detection.

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 80oC for 3 hours to release sialic acids. The released sialic acids are collected by ultra-filtration through a 3,000 NMWCO filter and derivatized with DMB. The fluorescent sialic acid derivatives are analyzed by reverse-phase HPLC with on-line fluorescence detection. The method allows detection of as little as 250 femtomol of individual species, and separates most of the known members of the sialic acid family. 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. Pretreatment under basic conditions to remove O-acetyl gropups may de desireable if one simply wants to the know the amount of Neu5Ac present.

Com-006. Sialic Acid Determination 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 80oC for 3 hours to release sialic acids. The released sialic acids are collected by ultra-filtration through a 3,000 NMWCO 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 acids 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.

Com-007. Anion Chromatography (phosphate/sulfate).

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. Electrochemical detection is used. The printout of the results shows the profile and sulfate and phosphate content expressed in nanomoles present in the volume injected. 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.

Linkage Analyis

Lin-001. GC-MS of PMAA derivatives. The starting material for this procedure should be purified glycopeptide, glycolipid or glycan. These are methylated according to the Ciucanu-Kerek or Hakomori methods as appropriate. Purification methods used include extraction, Sep-Pak cartridge, 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 the alcohols are derivatized to obtain partially methylated alditol acetates. Analysis of these products is done by GLC-EIMS, and identification achieved by using a combination of retention times (as compared to those of known standards analyzed under the same conditions) and EIMS patterns (mass spectra).

Mass Spectrometry

Mas-001. ESI-IT Mass Spectrometry (LCQ; direct infusion). The starting material for this procedure is typically glycopeptide, glycan or synthetic compound. Buffer and salt should be minimal. An autosampler is used to inject the sample into an open capillary which first directs the analyte to a UV detector and then into the LCQ mass spectrometer. Spectra are typically obtained in the postive ion mode. Detection limits in the low pMole range are typical. The printout includes the UV trace, the total ion current and the mass spectra obtained by summing the spectra collected after the injection and in the peak of the total ion current.

Miscellaneous Mis-001. Powerpoint presentation 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-002. Additional technical time.

There may be an additional charge if custom methods are requested or if methods need to be developed. This charge will not be imposed without prior consultation with the investigator.

Mis-003. Consultation.

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-004. Collect Fractions.

Given proper prior notification, members of the Glycotechnology Core will collect column chromatography fractions during any analysis requested (profile, composition).

Mis-005.

Collect Fractions with Scintillation Counting. Given proper prior notification, members of the Glycotechnology Core will collect column chromatography fractions directly into scintillation vials, add scintillation cocktail and subject the fractions to scintillation counting. The printout typically includes cpm and a measure of counting efficiency.

Mis-006.

Collect Fractions with Scintillation Counting of a Portion Only. Given proper prior notification, members of the Glycotechnology Core will collect column chromatography fractions, remove an aliquot to a scintillation vial, add scintillation cocktail and subject each to scintillation counting. The printout typically includes cpm and a measure of counting efficiency.

NOTE:

Most of the charges for the services provided by this Core are based on the assumption that the investigators provide an adequate sample, and are capable of interpreting the results themselves. Thus, the Core Director will not be involved in routine services, except to the extent of approving the samples for analysis and reviewing the results before they are provided to the investigator. If, for any reason, the sample does not allow routine analysis, and requires more than a half hour of the Core Director's time for interpretation and/or setting up of modified methods, Consultation Fees (GL0011) will be charged on an hourly basis. It should be noted that because of the large volume of requests from many sources, the availability of the Director's time for consultation may be limited. Priority will be given to projects originating from UCSD. A standing Advisory Committee will advise the Director as to which projects and systems can be pursued within the time available.