Mixed proteomes and the hunt for purity

By Kate McDonald
Monday, 23 June, 2008


It has been a hell of journey from a 17-year-old laboratory technician in New Zealand prodding away at snags and minced meat to becoming an integral part of the pioneering proteomics group at Macquarie University's Advanced Proteomics Analysis Facility, but that is the journey taken over less than two decades by Ben Herbert.

Now the director of the Proteomics Technology Centre of Expertise at the University of Technology, Sydney, Herbert is one of the Australia's leading experts in isoelectric focusing and 2D electrophoresis.

It certainly didn't start out that way. Herbert initially did a certificate in chemistry through Christchurch Polytechnic and was content enough as a laboratory analyst doing chemical analysis of fat content, water content and preservatives in food for NZ's then Department of Scientific and Industrial Research. A colleague by the name of Jeff Plowman, who is now with AgResearch, had a PhD in protein chemistry and was interested in doing species determination in food.

"I got to do some work with him as a technician and ultimately went onto that within a fairly short time, doing antibody-antigen tests for species in meat and then looking at isoelectric focusing to look at species in fish," he says. "We built up a big collection of fish from all of the different commercial species in New Zealand. That was fascinating stuff to me because I was 17 years old and it beat the hell out of testing fat in sausages. That was the real start for me in doing protein extractions and separations."

Herbert moved on to work with NZ's Wool Research Organisation, which allowed him to do a university diploma, where he worked with a small cell biology group looking at the strength of wool fibres. Here, amongst a lot of molecular biology, he started doing protein chemistry - generating one- and then two-dimensional electrophoresis of wool proteins.

"We bought the first system in New Zealand to do 2D gels with immobilised pH gradients (IPGs)," he says. "That was a big deal at the time and we published the first paper anyone ever did on 2D gels and wool proteins using IPGs."

That research took up five years, but in the meantime he had popped over to Australia for a workshop on protein chemistry at Macquarie University, where he just happened to meet Professor Keith Williams and his now very well-known group, which pioneered proteomics research in Australia. This group, including Andrew Gooley, Nicki Packer, Brad Walsh and Mark Molloy, amongst others, were surprised to find that the young Kiwi was quite comfortable running 2D gels, which they perceived to be reasonably difficult.

He ended up running a few workshops on 2D gels for Macquarie and was then invited to come over permanently to do his PhD. This was in 1995, when the Macquarie group was organising and lobbying for funds for what was to become the Australian Proteomics Analysis Facility (APAF), the first of its kind in Australia.

---PB--- Commercial links

The people behind APAF have all since gone on to bigger and better things. The core group left at the end of 1998 to set up the biotech company Proteome Systems, which is still going strong today, led by Jenny Harry. Some, like Mark Molloy, are now back in academia, as is Herbert. Others, like Brad Walsh, have set up their own private companies.

"All of the people in the group [at APAF] were being offered jobs elsewhere, and Keith knew that the key thing was to try to keep the group together," Herbert says. "We looked at getting a CRC and were defeated at the very last hurdle. At that point a lot of the groundwork had been done and we had so many commercial contacts that the idea was floated that we should put together a company, so we did. It started with four of us - me, Andrew, Nicki and Keith - and then Mark Wilkins came on board and then Jenny Harry, who was the last one of the founders."

Herbert admits it was a difficult time for all involved but APAF had been a somewhat commercial entity anyway. He still receives royalties for some of the technology he helped develop with Bio-Rad, including sample preparation kits and an isoelectric focusing instrument. He also helped with the initial development of Bio-Rad's IPGs.

There were two separate but linked goals in setting up Proteome Systems, he says. One was the continued development of the technology platform developed at APAF, which had the potential of being commercialised.

"The second aspect was to use that platform to look at diseases and apply it to biological and medical problems and either fund those ourselves or work with partners who wanted access to our technology platform. The company has really evolved into one that has a technology platform that it uses to do all of its proteomics research and effectively develop the outcomes of that into diagnostics."

Herbert worked with Proteome Systems for seven years before being lured back to the university sector by the University of Technology, Sydney. There is still an APAF/Proteome Systems link, however - Jenny Harry's sister Liz Harry is an associate professor at UTS' Institute for the Biotechnology of Infectious Diseases and was very keen to get proteomics started at the university.

Herbert now runs the Proteomics Technology Centre of Expertise, part of UTS's plan to establish a series of technologically focused core facilities.

"The university has been extremely supportive of the whole idea," he says. "We've had plenty of financial support from the university and have been able to buy most of the equipment that we've needed. The break in my link with Bio-Rad when I left Macquarie and up to now hadn't stopped me still talking to them, so when I joined UTS, Bio-Rad came on board as our industry partner. The uni saw that as a big plus, that we could have an industry partner. We act as a showcase facility for them and we do training courses, both here and overseas."

---PB--- Protein science conference

During his time at APAF, Herbert had the good fortune to meet and collaborate with a true pioneer of proteomics technology, Professor Pier Giorgio Righetti, then at the University of Verona. Righetti is also speaking at the Asia Oceania Human Proteome Organisation (AOHUPO)/Pacific Rim International Conference on Protein Science in Cairns in June.

In the world of proteomics, fractionation of complex samples has been one of the biggest technological breakthroughs of the last 15 years. At the time, Herbert thought fractionation by isoelectric point looked like an excellent way to break up those samples and run the fractions on narrow pH gradients.

"I had always been a huge fan of Righetti's work because he was so prolific and had been an inventor of so many of the technologies that were out there," Herbert says. "It turned out one of those was a liquid phase isoelectric focusing system called a multi-compartment electrolyser. I read about these things and decided that what it had been built to do was purify individual proteins to complete homogeneity for NMR or crystallography, but it seemed to me that you could equally use them to fractionate complex mixtures."

Herbert organised for Righetti to come out to Macquarie to speak at a meeting he and Brad Walsh were organising. From then on, Herbert and Righetti have worked in close collaboration and have published widely together, predominantly on 2D electrophoresis and isoelectric focusing.

At AOHUPO, Herbert will discuss, of all things, fungal infections. Herbert and his new team at UTS are working with Associate Professor Dee Carter from the University of Sydney, assisting them understand the proteomics of fungal infection, using Cryptococcus gattii as a model. C. gattii is not a common infection in humans, although there has been an outbreak in Vancouver recently, but it is most definitely a problem for koalas, as it lives in eucalyptus trees.

"Our real involvement in this is working with how to break open Crypto and get protein out of it. It's got an incredibly and extremely sticky capsule around the cell which is involved in the virulence of the organism. The capsule actually gets bigger when it is in a lung because that is a more stressful situation for the fungus to be in.

"It's been one of the most difficult organisms we've ever worked on, trying to figure out how to break it open and how to break the sticky interaction between the proteins and this capsule. What happens is if you are not very careful all of the proteins are lost and you actually just spin them out in the centrifuge and chuck them away, thinking it is cell debris."

Herbert's team has hit on an old-fashioned but seemingly forgotten method of breaking these interactions and preparing pure samples - salt. "I wanted to test which salts work the best. We've looked at monovalent and divalent cations and found that lithium chloride works better than anything else. I think that is because it is the smallest atom and therefore you have the highest charge density and you can get it into small areas. And we find it is a really good stripping agent, it gets in and dissociates protein from the capsule and we can then recover significant amounts of protein from Cryptococcus."

---PB--- Pure and unpure

A major topic he will be covering at AOHUPO is the challenge of mixed proteomes and his crusade for purity. His team has found that when cells are extracted from the homogenised lungs of infected animals, the strength of the Cryptococcus and its stickiness is actually quite useful.

"You can homogenise it and wash away all of the lung tissue and you are left with a really pure prep of Crypto, but even after repeated washing, if you then go in and strip that with lithium you then find a huge amount of lung and mouse proteins still stuck on the surface.

"People have to start to think purity doesn't exist anymore. You have this lovely tube full of lovely cells and you look at it down the microscope and you think you have washed it properly. Then you lay it on the gel and think there is nothing in it, but by putting lithium chloride on them and wash them in that, and then put them in the mass spectrometer, up comes huge numbers of mouse proteins.

"What we have learned from it is that lithium is a very powerful agent for disrupting interactions and we are now working with lithium as a stripping agent for membranes, for example. I have an honours student doing membrane protein work with lithium as a stripping agent instead of sodium. What I want to say to the audience up there is, don't get bogged down in dogma that something has been solved just because it has been published a hundred times. That doesn't mean it is right and you might find a better way."

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