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Techniques & Tools Proteomics, Liquid Chromatography, Mass Spectrometry, Technology

Perfecting Proteomic Separations

sponsored by Pharma Fluidics

How did you get involved in proteomics research?

Kris: I did my Master’s thesis in a proteomics lab in the mid-1990s, and have been in the field ever since. I became a professor at Ghent University in 2004 and a group leader at VIB in 2005. Proteomic technologies developed in our lab are made available to other researchers via our VIB Proteomics Core.

Francis: I’m a biomedical scientist by training. It was Kris who introduced me to proteomics – I did my PhD thesis in his lab. After a stint at the Pasteur Institute in Paris, working on bacterial pathogens, I returned to Ghent University and VIB in 2015. I manage the proteomics core facility – a spin-out from Kris’ lab. I also have a small research group “on the side,” which focuses on host–bacteria interactions using proteomics technologies.

What motivates you?

Francis: For me, it’s the strong belief that this technology is enabling real discoveries, as we witness on a weekly basis. There’s still so much to discover – novel interaction partners, novel protein modifications, novel biology!

Kris: I also enjoy the opportunity to work with scientists in a variety of disciplines, from plant biologists to clinical scientists.

What recent trends have you noticed in the daily routine of the proteomics center?

Francis: Proteomics technology as a whole has matured, with a very rapid evolution of mass spectrometry instruments, separation instruments, and supporting technologies. It’s a very different landscape compared with just a decade ago. As a consequence, some proteomic analyses (such as shotgun analysis for biological samples and mapping protein interaction after affinity purification) have become routine. Of course, there are still many novel kinds of analyses, which are still very much dependent on the expertise of single labs or even single people.

Kris: There are certainly some exciting new techniques that allow us to focus on aspects of a protein that we could not study some years ago. In my lab, we invent or trial such techniques to master them and then transfer them back to our Proteomics Core as a new tool for the wider user community.

What are the biggest limitations in proteomics?

Francis: With shotgun analysis, we can only routinely measure about half of the proteins expressed in the average mammalian cell. And that is not taking into account the millions of different proteoforms that could also exist. The question is: do they all have meaning? To me though, it’s not a limitation – it’s a challenge.

Will the resolving power of mass spectrometers eventually eliminate the need for separation technology?

Kris: The short and simple answer is no. We will always need resolving power, be it LC or other types of chromatography. It is simple mathematics: there could be hundreds of thousands of different peptides in a sample – and it’s impossible to identify that number of molecules without separations.  

Can improvements in separation technology help overcome the challenges facing proteomics?

Francis: In my view, improving separations is key. Mass spectrometry has seen huge advances over the past 10 years, for example, Orbitrap technology and hybrid machines. To help mass spectrometry reach its full potential, we need to see advances in fractionation too – smaller peaks, more resolution, and higher peak capacities can only increase the overall performance of the analysis. To that end, we have been excited to trial the new micro-pillar array chromatography (µPAC) columns from Pharmafluidics.

We’re looking forward to finding out what else we can do that we haven’t been able to do before!
How can µPAC technology improve proteomic analyses?

Francis: Personally, I think it has the potential to revolutionize the field of mass spectrometry-based proteomics. It is the type of technology that speaks for itself – you can look inside the column, see the perfectly ordered structure, and understand exactly how it increases separation power.

Kris: In comparison with traditional columns, µPAC columns result in more sensitive analysis, so you need less material to identify the same number of proteins. Another positive of the µPAC columns is the ease of use – it’s very quick and efficient to put a µPAC column in front of your mass spectrometer. The back pressure is very low, meaning that you can work with long columns that allow greater sensitivity.

The µPAC columns have been designed by scientists who have been in the field of chromatography for decades, so we felt confident in agreeing to collaborate with them to implement the technology.

How have you applied µPAC technology?

Kris: So far, we have been evaluating them for routine shotgun analysis, where we try to gain a total protein profile. Compared with traditional columns, we see an overall increase in identifications, both on the peptide and the protein level, which results from the improved LC parameters that you can readily see just by looking at the chromatograms.

Beyond routine analyses, we are excited to try out µPAC technology as part of a wide range of proteomic methods. These columns have already made it possible to do incredibly long runs of eight or ten hours – something that was previous unheard of. We’re looking forward to finding out what else we can do that we haven’t been able to do before!

Will you be implementing µPAC into daily operation?

Kris: Yes, that is on the horizon. We are just waiting for some final aspects, such as a compatible trapping column, but we hope it won’t take longer than a few weeks.

In the longer term, where do you expect µPAC technology to have the greatest impact?

Francis: One of the biggest gains the company reports is in analyzing minute amounts of sample – µPAC boosts the performance of the mass spectrometer by keeping peptide concentrations undiluted and high prior to ionization. This particular aspect could be a game changer for analysis of small samples; for example, in biomarker discovery.

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