by Laura Lane
Compared to purifying and fractionating protein samples, the seeming impossibility of finding a needle in a haystack turns out to be quite easy. Burn away the hay. Use a metal detector. Eureka! Let the sewing begin.
The haystack idiom best suits the task of isolating specific proteins from biofluids, cell and tissue lysates, or other types of samples. All the proteins consist of the same 20 amino acids. And, you’re walking on needles trying to preserve the integrity of the proteins. No purging flames here—you must choose the conditions very carefully. While certain buffers and solutions may do the trick in one scenario, the same protocol could be disastrous in another. Some proteins can withstand the battering of centrifugation; others can’t.
“The challenge with protein sample preparation is that there’s no silver bullet; there’s no one-size-fits-all solution,” says Mary Grace Brubacher, marketing manager of expression proteomics at Bio-Rad. “It’s due to the complex nature of proteins.”
For decades now, molecular weight and isoelectric point have served as key factors in separating proteins with gels and columns. While size and charge continue to reign, suppliers and researchers have modified and improved separation technologies to tackle the challenges of drug discovery and the proteomics movement.
“People are using a real mix of techniques from very basic to quite sophisticated depending on what they need to do with the protein, and it’s important it get it right from the start to achieve good results” says Penny Owen, segment lead of the consumables division at GE Healthcare Life Sciences.
Better, Stronger, Faster
Several factors play into the protein sample preparation techniques that researchers choose; however, ease, convenience, and efficiency are always part of the picture. Companies have responded with kits, packages, and devices that maximize hands-off time and minimize fuss. Saving time not only saves money, but also the proteins themselves.
That would be welcome news to researchers like Daifeng Jiang, who works in a lab that has spent the last decade trying to recover transcription factors. A research assistant professor at the University of Texas at San Antonio, Jiang and her colleagues have so far developed several techniques to purify eight transcription factors, such as AP1, Sp1, C/EBP, Gal4, USF-2, and the lac repressor.
“It’s a challenge,” Jiang says. “We need to keep the activity of the protein, so we have to be very careful of what we do with it.”
One key, says Richard Friday, marketing director for chromatography at GE Healthcare Life Sciences, is to “minimize the time that the protein is exposed to non-native conditions. The quicker they can do it, the more chance they have of maintaining the protein’s [biological] activity.”
Then again, you may be completely content with studying peptides of digested proteins. This would not bode well if you had hoped to perform a biological assay. The bottom line: The strategy you choose should reflect what you do next.
“Quality sample preparation is one of the major keys to successful proteomics experiments. If you don’t get all the steps right, you’ll hinder the downstream performance and measurements,” says Mike MacCoss, associate professor of genome sciences at the University of Washington.
Search, Rescue, Recovery
If you’d like to keep the proteins intact throughout the preparation process, you now have several options. One of the latest is the Gelfree 8100 Fractionation System by Protein Discovery.
Prior to its introduction in June 2009, MacCoss had been analyzing proteins by shotgun proteomics, which involves digesting a mixture of proteins into peptides that are analyzed directly by liquid chromatography-tandem mass spectrometry. Unfortunately, without information about the intact protein, interpretation of the data can be complicated. For example, you’re unable to determine if the detected peptides came from a protein that was truncated or intact. Now, MacCoss separates complex protein mixtures in molecular weight fractions using the Gelfree system, and performs shotgun proteomics on the individual fractions. “This strategy simplifies data interpretation of shotgun proteomics because you maintain information about the molecular weights of the intact proteins,” he says. “It tells you a lot more.”
GELFREE is an acronym that stands for gel eluted liquid fraction entrapment electrophoresis. The device relies on the same principles of one-dimensional gel electrophoresis, says Chuck Witkowski, president and CEO of Protein Discovery. Proteins separate according to their electrophoretic mobility in precast gel tubes. The proteins continue to migrate to the end of the tube, out of the gel, and into a liquid solution, where you can collect that fraction with a pipette.
Use the device’s touch screen to choose from several preset fractionation protocols or to define your own custom protocol. Additionally, up to eight samples can be simultaneously processed in parallel.
“Rather than cutting a band out of a 1D gel, you simply elute the protein out of the gel and recover the liquid with a pipette,” Witkowski says. “The format allows you to recover proteins intact with higher yield.”
Bio-Rad’s Rotofor product is also designed for solution phase isoelectric focusing and recovering the protein in an intact state. With a solution-based pH gradient, you can collect fractions and use them for downstream analysis, including those that assess the protein’s activity.
“You can keep the protein in its active state for biological assays,” says Kumar Bala, marketing manager at Bio-Rad. The company also offers the technology as theMicroRotofor, which aids the processing of small sample volumes with reduced amount of proteins.
Low Down On Low Abundance
Of course, the first goal is to find a way to actually capture the protein—a high order when the mission calls for low abundance proteins.
The Gelfree could also be the solution. The specialty gel tube allows you to “load at least five times more total protein than a 1D gel,” he says. “With higher load capacity and higher recovery for each fraction, that means the likelihood of detecting low abundance proteins is higher.”
Another strategy is to miniaturize previously developed technologies that clear away high abundance proteins, thereby enriching for low abundance proteins.
Last year, the company introduced a small capacity version of ProteoMiner. The product hinges on a combinatorial library of hexapeptides, which are bound to beads. Because of their great diversity, the hexapeptides bind to proteins traveling through the bead column. High abundance proteins quickly saturate their complement of hexapeptides, while excess proteins flow through. Low abundance proteins continue to bind. During elution, you capture the low abundance proteins while reducing the high abundance ones.
“The beauty of ProteoMiner is that it uses a library of peptides instead of antibodies,” says Kate Smith, product manager for sample prep at Bio-Rad. “Because of that you’re not limited to serum as the only sample type nor are you limited to a particular species as with immunodepletion.”
GE Healthcare also offers solutions for capturing low abundance proteins. They include Protein A and Protein G magnetic beads that can be scaled to different sample volumes, and Nickel Sepharose™ beads, which come in a range of formats for different sample sizes.
“You can scale up or scale down with confidence,” GE Healthcare’s Owen says.
Even then, you want to ensure that your hard work isn’t sabotaged. All too often, the perpetrators are the very plates and other plastics that come into contact with your sample.
“Conventional plastics for biology purposes get instantly saturated with biomolecules,” says Jesse Cassidy, product manager of sample prep products at Eppendorf, explaining that this could mean up to 90% of the proteins in a sample. “Unlike DNA for which you can easily make more copies, making more copies of your protein of interest is time consuming and costly.”
Eppendorf developed the LoBind surface for plastic consumables to prevent such loss. Contrary to silicone-based coatings that tend to leach into the sample, the LoBind technology consists of the proprietary material that’s actually combined with ultrapure polypropylene. The LoBind line includes microcentrifuge tubes, deep-well plates, and 96-well and 384-well microtiter plates.
The company also offers pipette tips that are designed to minimize sample loss and improve accuracy.
Today’s advanced capabilities open many doors to protein exploration. Still, methods to isolate certain membrane proteins or those that are very acidic or basic remain elusive. With such an unwieldy collection of proteins that exist, “there are still unmet needs in protein sample preparation,” Bio-Rad’s Brubacher says. “You can expect suppliers to introduce more solutions in the near future.”