Currently, my project at work is focused on the analysis of carbohydrates in glycoproteins.  This analysis includes the determination of sugar composition, sialic acid content and oligosaccharide structures.

Why do we have to perform these tests on proteins?  The majority of proteins are in the form of glycoproteins.   Oligosaccharides play  critical biological roles in cell-cell interaction, embryonic development, tissue organization and morphogenesis.  In addition, they also play an important role in protecting and stabilizing of proteins.  It is therefore necessary to examine the carbohydrate portion in proteins in order to have a better understanding of how they effect the biological roles of proteins.   Herein I will describe briefly how these tests are done.

In sugar composition, the percentage of each monosaccharide is determined.  First of all, glycoprotein is subjected under an acidic hydrolysis (TFA) to cleave monosaccharides off the protein.  These are then labeled with a fluorescent tag, anthranilic acid (AA).  The labeled monosaccharides are then separated using RP- HPLC with a fluorescence detector.  A set of standard monosaccharides is also subjected under the same condition.  This is used to generate a standard curve from which the content of each monosaccharide in the protein is determined.  Using this technique, as little as nmol to pmol of protein is required for the whole test.

Sialic acid or N-acetylneuraminic acid (NeuNAc) is a ketose- type sugar.  It is known that NeuNAc has some impacts in the pharmacokinetic (PK) property of protein.   It is therefore interesting to find out what is the correlation between NeuNAc content of a protein and its PK value.  NeuNAc is cleaved from protein under a mild acidic condition (NaHSO₄).  It is then labeled with o-phenylenediamine dihydrochloride (OPD 2HCl), and separated using RP -HPLC with either a UV or fluorescence detector.  For this test, sialyllactose, which upon hydrolysis releases NeuNAc, is used to generate a standard curve.

The determination of oligosaccharide structures is very complicated due to various possibilities of how one monosaccharide is connected to the others, plus its anomericity.   Most of the proteins I am dealing with are N-glycosylated.  Therefore the intact oligosaccharide is released from the protein using PNGase F.  The protein is first digested with trypsin/chymotrypsin prior to treating with PNGase F to enhance the extent of deglycosylation as well as the reliability of the enzyme.  The resulting oligosaccharides are then tagged with AA, separated using NP-AEC with a fluorescence detector.  The separation is based on the charge of the sugar chain which depends on the number of NeuNAc attached to it (e.g. 0-4 NeuNAc /chain).  The collected oligosaccharide is then subjected to MS for mass determination.  By comparing with the masses in the database, the structure of the oligosaccharide is partially elucidated.  Yet exoglycosidase must be used in order to distinguish Man from Gal since they have the same MW.  By now only some structures are solved.  I still have a long way to go before the whole sugar map is solved...

Back to My Research & Job