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New technique for specifying location of sugar chains on glycoproteins

Date: 23.11.2009 

Researchers have previously been able to analyse which sugar structures are to be found on certain proteins, but not exactly where on the protein they are positioned. This is now possible thanks to a new technique developed at the Sahlgrenska Academy at the University of Gothenburg, Sweden. The technique entails preparing samples in a new way and is a development of applied mass spectrometry. Presented in the latest issue of renowned journal Nature Methods, the technique will enable medical researchers to study the mechanisms behind diseases in more detail and, with luck, find new ways of treating them.

Picture: schematic representation of N-linked protein glycosylation at asparagine (Asn) residues in glycoproteins

"We were analysing cerebrospinal fluid from healthy subjects and could see that many proteins had sugar structures previously unknown to us," says Jonas Nilsson, a researcher at the Department of Clinical Chemistry and Transfusion Medicine at the Sahlgrenska Academy. "We know that some of these proteins play a role in diseases such as Alzheimer's disease, and now it's possible to study whether faults in these sugar structures are responsible for the development of the disease."
Proteomics is the large-scale study of proteins, particularly their structures and functions. There are more than 20,000 proteins in the human body. These proteins ensure that the instructions from the genes are carried out. Proteins will vary with time and distinct requirements, or stresses, that a cell or organism undergoes. Any one protein can undergo a wide range of post-translational modifications. Therefore a proteomics study can become quite complex very quickly, even if the object of the study is very restricted.

Around half of proteins in human body undergo glycosylation, the process that links saccharides with proteins. This is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation. Glycoproteins serve a variety of structural and functional roles. The polysaccharide chains alias glycans attached to the target proteins serve various functions. For instance, some proteins do not fold correctly unless they are glycosylated first. In some cases the unglycosylated protein degrades quickly. These sugar structures mean that the protein can be recognised by other proteins, too. Some of these structures can act as a locking mechanism when proteins bind to cells and other proteins, which is employed mainly by cells of the immune system. Sugar structures on the surface of cells determine, among other things, which blood group we belong to.

There are two major classes of glycans: N-linked glycans attached to a nitrogen of asparagine or arginine side chains, O-linked glycans attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains, or to oxygens on lipids such as ceramide. Further C-linked glycans or phospho-glycans are produced.
For proteomics study generally mass spectrometry with matrix-assisted laser desorption/ionization (MALDI MS) is using for rapid determination of proteins. Mass spectrometry is an analytical method which can be used to determine the mass of positive or negative ions. The method can also be used to identify large molecules such as proteins and measure how much of a particular protein a sample contains.

"Sugar structures often play an important role in how a cell or protein functions and how it affects different systems in the body," says Nilsson. "Being able to study them in more detail is a major step forward for biomedical research."
The chains of sugars in these structures are attached to the proteins at only one end. The new technique entails attaching a plastic bead to the loose end of these chains and separating the sugared proteins from those that do not have sugar structures. The proteins are then trypsinized and the sugar chain is released from the plastic bead, leaving the sugar chain attached to a chunk of protein known as a peptide. The researchers can then study the sugar structure on the peptide and see which protein the peptide belonged to and where on the protein it sat. Mass spectrometric fragment analysis allowed identification of glycan structures, and additional fragmentation of deglycosylated ions yielded peptide sequence information, which allowed glycan attachment site and protein identification. We identified 36 N-linked and 44 O-linked glycosylation sites on glycoproteins from human cerebrospinal fluid.

Author: Dagmar Smětalová





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