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Capillary Electrophoresis-Mass Spectrometry – The Coming of Age for a Powerful Technique

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“CE-MS is a powerful analytical technology that combines a separation technique – capillary electrophoresis – with mass spectrometry as a detector,” explains Dr Rob Haselberg, Senior Postdoctoral Researcher at Vrije Universiteit Amsterdam.

The technique separates compounds based on their mass and charge – including small molecules, metabolites, drugs, peptides and proteins – and can identify and quantify them. A widely-used alternative is liquid chromatography-mass spectrometry (LC-MS).

“Until a few years ago, LC-MS was not so good at separating proteins. While it’s improving, CE still has the upper hand in that,” says Haselberg. “But there are many studies on CE and LC that show they are actually very complementary – and that’s important so you can verify whether you’re missing things.”

Benefits of CE-MS

“The advantages of CE-MS are that it’s fast, it has high resolution, and it’s also robust and reproducible,” says Professor Harald Mischak, Director of Mosaiques Diagnostics. 

“On the other hand, there are shortcomings. One is it has a limited sample volume loading capacity. This isn’t a problem for routine analysis, but it can be if you want to perform sequencing – so for this, we’d need to use an alternative method,” he adds.

However, the need for tiny sample volumes can be helpful sometimes – for example, when work involves precious patient samples.

“Another disadvantage is that we’re restricted to short run times, which enables us to analyse 5-6,000 compounds – and that’s the limit,” he adds. “However, I don’t see this as a problem, at least for how I use it – because if you were able to analyse more, then you would need a few hundred samples to really investigate the low abundance molecules in depth - which often isn’t practical for patient samples.”

By far the biggest advantage of CE-MS is its power. It can detect subtle protein variations that would be missed by most other technologies.

Erythropoietin (EPO), a protein used to treat patients with poorly oxygenated blood, provides one such example. Synthetic EPO is taken by some cyclists as a performance-enhancing drug (although recent evidence casts a doubt over its benefits), but also occurs naturally in the blood. It can, therefore, prove challenging for anti-doping agencies to pinpoint the synthetic version.

“EPO is considered a pure drug, but it does have glycosylation – so variants with sugar units attached. It’s striking what CE-MS can do, with just one single analysis we can identify around 300 variants,” enthuses Haselberg. “LC is getting better, but it’s still far away from what this can do.”

CE-MS steps into the spotlight

The last decade has witnessed a revival in technological developments for CE-MS, while at the same time moving away from being a purely academic tool to one increasingly being adopted by industrial scientists.

“Until a few years ago there was only one commercial supplier offering a technology solution, meaning little progression. But more recently, there have been new players entering the market with new technologies. This started a few years ago and will get better and better, making CE-MS more sensitive and more robust,” says Haselberg.

“With these advances in technology, you’ll see industrial scientists embracing it more and more, which means that you have complementary techniques for drug quality control, for example. For chiral separations, it was - and still is - a very big player, as chirality and CE go hand in hand,” says Haselberg.

Others are making impressive strides towards miniaturising the technology, with the development of microfluidic CE-MS.

“From what I’ve seen, miniaturisation will enable a lot of high-throughput work, without compromising the level of information you get out,” says Haselberg. “This will mean any process you want to monitor becomes more cost-effective.” 

Benefits for patients

Detecting biomarkers in body fluids is currently a huge area of research that is paving the way towards a future of personalised treatment, potentially offering a host of patient benefits.

“We’re looking to use CE-MS to identify biomarkers that enable the early detection of kidney disease, cardiovascular disease, bladder or prostate cancer – or for monitoring patients for drug response,” says Mischak. “The analytes we are investigating are urinary peptides, and it works very well and is very reproducible.”

“In fact, it’s already being used in clinical applications. I have a couple of tests on the market that, for example, are being used to assess patients at the early stages of diabetic nephropathy, for patients after a stem cell transplantation to identify graft versus host disease, and for patients with cholangiocarcinoma,” he adds. 

The technique has huge potential for earlier identification of disease. For some illnesses, detecting initial changes in a person’s body fluids at the molecular level opens the possibility of intervening to stop them from developing the full-blown condition.

“We need to shift away from our current repair medicine, where we are trying to fix the problem once the patient is sick, towards prevention,” says Mischak.

An exciting example that illustrates this is the PRIORITY clinical trial involving people with Type II diabetes across Europe.

“We’re using CE-MS to detect diabetic neuropathy early – before the disease is evident,” explains Mischak. And we’re then performing an intervention with spironolactone drug treatment or placebo – aiming to demonstrate an ultimate benefit, preventing patients from developing the disease.”

A myriad of uses

It’s not just biomarker detection where CE-MS can help. Another example is in anti-doping analysis, where it can help distinguish proteins used by athletes to cheat, such as EPO or growth hormone. 

The biopharmaceutical industry is also starting to wake up to its potential. For instance, it can help characterise biological drugs, such as antibodies used as cancer treatments, identifying post-translational modifications that aren’t possible with any other technology.

By combining structural analyses with activity assays, CE-MS is a useful tool for identifying active compounds within complex mixtures, which could be developed into new drugs.

“We’re collaborating with industry on a project looking to correlate certain biological activities to specific compounds within snake venoms,” says Haselberg. “Some snakes prevent blood clotting so the victim bleeds to death, but this is also a target for lowering blood pressure if you dose it in the right way.”

“As you can imagine, you don’t want to milk a snake every time you want to do an analysis, so CE-MS helps as we only need a small sample to look at lots of things,” he adds.

A bright future

Over the past few decades, CE-MS has remained out of the limelight. However, there is now revived momentum to exploit some of the unique advantages it can offer.

These include scientists hoping to discover robust clinical biomarkers, which will propel us into a new era of personalised medicine.

“I think CE-MS will be used as a routine clinical tool,” says Mischak. “Of course, there’s a need for some additional developments, but there’s a big future for it.”