Characterising glycans not just the icing on the cake

Oligosaccharides are the third-largest solid component in human breast milk, following lactose and lipids, but no one is completely sure of what they are there to do. They seem to have no nutritional value for infants but if they are there in such abundance there will definitely be a good reason.

One popular theory is that they stimulate the innate immune system in newborns and have a pro-biotic role in providing food for essential intestinal microflora. This sounds logical but to find out more has been difficult, partly because of the extreme complexity in profiling and characterising these types of sugars.

Taking a closer look at oligosaccharides or glycans seems to be giving rise to yet another in 'omics' families, this time glycomics. The field falls in between proteomics and metabolomics but is obviously related to both. Professor Carlito Lebrilla, a professor of chemistry at UCD, has been taking a very close look indeed at glycomics as part of a wider research program called Foods for Life.

"Foods for Life is a new paradigm in health," Lebrillo told ALS on a recent trip to Australia, where he spoke at the Lorne proteomics conference.

"The old paradigm was that you get sick and then we'll try to fix it. The new paradigm is to monitor health and to keep someone healthy. How do you keep someone healthy? Diet is obviously important, so the question then is, what's the perfect food?"

Any number of food companies or nutritionists could proffer a few examples, but human breast milk has to be up there. For Lebrillo, the question is, what is it about human milk that makes it the perfect food for the infant.

"It turns out that there are a lot of components of human milk that have anti-cancer properties, anti-microbial, and people are just now trying to figure out what it is so we can then emulate it in what we eat," he said.

"It takes a lot of energy to produce oligosaccharides, [energy] that is taken away from the mother. So there has to be some role. One theory is that it's food for microflora, gut bacteria.

"We are monitoring the milk oligosaccharide and what happens to it during lactation. The second part of the project is that we are feeding it to gut bacteria and so we have a couple of papers out on how do you profile and how do you characterise oligosaccharides, which is a hard thing to do."

This is where the partnership with Agilent Technologies comes in. Lebrilla met Dr Rudy Grimm, proteomics and metabolomics market development manager for Agilent's Life Sciences and Chemical Analysis (LSCA) group, at a conference in Seoul three years ago. Lebrilla gave a presentation on his glycan research and Grimm was giving a presentation on high performance liquid chromatography chip/mass spectrometry (HPLC/MS) technology.

"We very quickly figured out that it would be fantastic to collaborate and come up with a new HPLC chip that is capable of separating all of the glycans that are present in biological samples," Grimm said.

"Carlito gave us some good advice about what materials we should use and depict that in chips and to create new chips. We then came up with a solution that really revolutionised the entire field of glycan analysis."

Lebrilla said the team has come up with a chip laced with activated charcoal that enhances and diminishes some of the interactions between the oligosaccharides and the graphite surface. "That makes some of the oligosaccharides travel a little faster and so it separates them and by combining that with mass spectrometry we get a profile that in the past required a lot of material and a long time to separate."

Grimm said a major difference was that, in the past, researchers have required a large amount of samples to separate.

"People had big problems with glycan separations in the capillary at the nano-scale and this is nano HPLC in an integrated chip - we are talking about glycan separations at 75 or 50 micron chip columns, so this means that you only need tiny amounts of samples to analyse.

"Even if you have large amounts available you can do lots of separations. Just from the basic chip layout we have developed a dead-volume-free system so we hardly have any dispersion in the separation system and that will lead to increased resolution and sensitivity and that's really ultimately where the power is."

Following the separation of the mixtures, time-of-flight mass spectrometry (TOF MS) is used to characterise each oligosaccharide with a mass accuracy of two parts per million. This level of accuracy is important in being able to deduce the glycan structures, Grimm said. Tandem mass spectrometry is then used to determine the structure of each individual oligosaccharide.

Human milk genome

The collaboration is also part of a wider picture, in which UCD is working on the Human Milk Genome Project. The new chip-based technique is capable of separating around 2-3000 different glycan compounds, including their isomeric structures, which have huge importance in understanding the morphology of bacteriophages that attack lactic acid bacteria used in fermentation of dairy products.

Another major project Lebrilla will work on is looking systematically for glycan-based biomarkers for different cancers, particularly ovarian cancer.

Last year, Lebrilla and colleagues identified 15 biological markers that appear to be present in the blood of patients with ovarian cancer, but not healthy women, and that were also produced by cancer cells in the lab. The project studied the oligosaccharides attached to proteins secreted by the cancer cells, stripping the oligosaccharides from the proteins and profiling the mixture.

Results of that study were published in the July 7, 2006 issue of the Journal of Proteome Research. The group plans to conduct larger clinical studies to test their findings but also wants to determine how the pattern of markers develops and changes in cancer patients, and whether it could be developed into a diagnostic test for ovarian cancer to use alongside other methods such as ultrasound.

Lebrilla and his colleagues have established a company, Glycometrix, to develop the patented technology.

And studying glycans may very well lead to biomarkers that actually have real value. According to Grimm, one of the biggest problems with proteomics is that in the last couple of years about 2700 biomarkers have been published but only seven received approval from the FDA, mainly because most failed in the validation stage.

"I feel confident that with people like Carlito, who is designing his experiments very carefully, that we can come up with more glycan-based biomarkers," he said. "And then we need to look at lipids and glyco-proteins.

"What we will do with our new glycan chip-mass spec technology is systematically check all cancers and re-profile them for glycan biomarkers - that's the project that's starting now. We are pretty confident that we will discover new glycan biomarkers and we are planning to do the validation of those with the glycan chip in conjunction with our new triple quadrupole mass spectrometer."