by Alan Doucette, Chief Scientist

I’m often asked how to be successful in research, and I share one simple piece of advice: Nothing ever works the first time. So, if you think about it, the key to success is quite simple – what are you going to try next?

As an analytical chemistry professor at Dalhousie University (Halifax, Canada), I feel like I chose the perfect career. My research area is in the field of proteomics, looking to improve the process of protein characterization through mass spectrometry. The mantra in my lab is “Every day we face a new problem. It’s never a dull day.” I embrace the challenges of a research lab, especially when it comes to helping shape the minds of graduate students. I’m most comfortable while working ‘in the trenches’, troubleshooting and tinkering with one of the lab instruments, including those that I’ve designed.

You see, designing a new instrument or device is just an approach to problem-solving – the right tool for the right job, they say. But if the tool doesn’t exist? Simple – you build it yourself. But how do you know what to make, if it hasn’t even been made yet? Well, that’s where the real ‘research’ comes in. You can’t fix a problem you don’t yet understand.

A bit of history…

In this case, the problem to understand was an old one – more than a hundred years old. Protein precipitation is a classic approach to isolate and concentrate proteins. Organic solvent precipitation was first studied in the early 20th century as a means of investigating the composition of serum. It gained particular importance after Edwin J. Cohn perfected a technique to enrich albumin protein from plasma. The Cohn process is a juggling act between several variables including the amount of organic solvent (ethanol), the pH, and the ionic strength of the solution. The resulting powdered albumin fraction served as an important blood substitute in World War II, as it could be easily reconstituted and administered to wounded soldiers on the field.

Fast forward about five or six decades, where precipitation was just one of many approaches to isolate proteins from complex mixtures. Chromatography, electrophoresis, and membrane filtration all proved reliable approaches to recover proteins, and became favored tools to prepare samples for analysis through mass spectrometry.
And therein lied the problem: precipitation just wasn’t RELIABLE.

In addition to all the hassle and mess of trying to isolate miniscule sample pellets through painfully difficult pipetting, precipitation had a reputation for inconsistency. Some days it worked, and some days it didn’t. Yields were highly variable, and the net result was that multiple proteins seemed to just go missing – in other words, the proteins could not be effectively precipitated.

But why is that? That’s the question I asked. Especially since it seemed like such a promising technique. Was it simply that newer techniques proved to be better? Or that something was being overlooked in the precipitation process? The answer to solvent-based protein precipitation turned out to be extremely simple – add a pinch of salt.

Some insight on how solvent-based precipitation works

In water, proteins look basically like tangled balls of spaghetti. They may look disorganized, but their folding is a result of an impressive tug-of-war equilibrium between various chemical entities on the proteins, especially surface charge, and the water molecules surrounding them. But what happens when you take away the water? In organic solvent, opposing charges become magnified, because there is no polar solvent in the way to shield the electrostatic charges. Like a sock stuck to a sweater out of the dryer, the positive residues on one protein combine with negative residues on another protein, resulting in a cascade clumping of sample – a precipitate.

So where does the salt come in? What I discovered is that the addition of organic solvent alone is not sufficient to induce precipitation. In fact, all proteins remain happily dissolved in solution, even as the organic solvent is ramped to a maximum. But as soon as a pinch of salt is added (say about 10 to 100 millimolar), all these proteins immediately crash out of solution. Why? Salt is just a stand-in for ions – any ionic species will do the trick. When the water content is lowered (by adding organic solvent), the force between opposing charges is enhanced. And so too is the force between salt ions and protein charged. Most people are familiar with isoelectric precipitation – that proteins are least soluble at a pH that cancels out their charge. Well, the same applies here. In organic solvent, the salt ions attract to proteins and cancel out the charge. No charge = no reason to stay soluble. Hydrophobic forces take over and proteins crash out of solution. Seems rather obvious now, but isn’t everything in hindsight?

Better tools for better science.

Knowing that precipitation can easily be used for reliable recovery, I also wanted to make it simple. I envisioned a device where protein solution, containing all their impurities, combined with organic solvent, and with the push of a button, purified protein is separated from the solvent. I assumed we could buy some type of device to do just that but quickly realized no such tool had yet been made. So I made one.

Well, I actually made several. The first 3D-printed prototypes were crude, but functioned to prove the concept. It took a while to assemble the right team of people, in the form of Proteoform Scientific, and refine the product into what it is today – the ProTrap XG.

Purity, yield, simple.

The ProTrap XG is a disposable, two-stage filter cartridge designed to automate the process of protein precipitation. The upper and lower components of the plastic device screw together just like a cap on a tiny tube of toothpaste. Easy to use, proteins are rapidly purified through the process of precipitation. The end result is consistent recovery of over 95%, together with over 99% purity. All you need is a benchtop centrifuge to make it work.

What’s next?

I am already looking for solutions to the next problems. Like integrating the ProTrap XG into complete top-down and bottom-up workflows. My research team is currently optimizing approaches for integrated protein digestion in high throughput format. The kinetics of precipitation have recently been worked out (here’s a teaser – it’s faster than you think). And I am looking at ways to scale the precipitation throughput in a multiplexed format. I continue to innovate around challenges in the lab, looking for simple solutions to complex problems, especially those that facilitate proteome analysis for all.