Improvements in Liquid Chromatography
Wolfgang Peti, PhD, professor in the Department of Chemistry and Biochemistry at the University of Arizona, talks to contributing editor Tanuja Koppal, PhD, about the recent developments in chromatography techniques.
Wolfgang Peti, PhD, professor in the Department of Chemistry and Biochemistry at the University of Arizona, talks to contributing editor Tanuja Koppal, PhD, about the recent developments in chromatography techniques. He talks about how chromatography has now become more modular, automated, high throughput, and user friendly, which has allowed him to run samples for protein purification in an unattended, flexible setting. While innovations in instrumentation are important, integration and ease of use of the software tools and customer service remain important criteria in deciding which equipment to buy.
Q: What are some of the recent improvements you have seen in chromatography techniques?
A: We have been using chromatography for the past 25 years, and the main thrust of our work is to use the structure and dynamics of proteins to understand their biological function. For doing that work, we need pure proteins, peptides, or protein complexes. Chromatography gives us access to materials to perform experiments that can provide information on protein-protein and protein-nucleotide interactions, or to solve the structure of these proteins and understand the biology that is involved. In the past couple of years, chromatography has become much more user friendly and the software has improved greatly, which allow for higher throughput and automation. We can also do a lot of experiments unattended, at least the routine steps, by writing methods implemented in the modern software, setting up the workflow, and letting the system run unattended. There is a lot of routine work in protein purification, so this is an important feature that chromatography now offers. Automation has become much more powerful, and it is easier to program methods using the new software tools, which has, in turn, improved the throughput.
Q: Are there other alternatives to chromatography when it comes to protein separation and purification?
A: We use only liquid chromatography to purify proteins that can be expressed in E. coli, insect, or human cells. We lyse the cells and then extract and purify the protein of interest. Precipitation is a very crude way to purify proteins. [We] can purify the protein using affinity tags, which is a high-affinity, high-specificity method that is most commonly used. Or [we] can purify the protein away from other proteins and/or cellular components using liquid chromatography separation, based on intrinsic properties such as size, hydrophilicity, or the charge of the protein of interest. Liquid chromatography is really the workhorse for every molecular or structural biologist and biochemist looking to purify proteins.
Q: How do these improvements in chromatography translate to changes in the workflow?
A: The innovations in the instrumentation for chromatography lie in the modularity, which gives you the freedom to change things in the setup. Previously when you bought chromatography instruments, it was hard to change anything. Now it’s easy to change, upgrade, and integrate instruments, which gives you a lot of flexibility. Furthermore, chromatography vendors realize that there are a lot of companies that supply specialized detectors and other equipment that they cannot offer. Hence, they offer solutions that can integrate these different pieces of equipment easily and rapidly into the existing chromatography workflow. Modularity has made integration possible, and maintenance on these instruments has also become fast and easy. That’s become an important feature because it minimizes downtime on these expensive instruments. It’s become effortless to write methods with the software, which makes automation easy as well. Chromatography is no longer a technique that can be used efficiently by only specialists.
Q: Can you elaborate further on how you have increased throughput in protein purification using chromatography?
A: In protein purification, you can’t run multiple samples at the same time since you have to purify one protein at a time. However, you can have many columns for purification set up back-to-back to run on the same system. Previously, the software for doing this would not allow for such a setup. But now, as long as you have the column- switching mode on your instrument and air sensors in your columns, [this] can be done very easily, and you can run the samples unattended overnight. When you come in the next morning, all the proteins are collected in fraction collectors. It’s not a fast technique, but with these new features, you can run two or three samples, instead of only one, in one day. Sometimes, when methods get very complicated with different columns to use and different buffers to mix, things don’t always go right the first time. It’s important not to get frustrated and to remember that once you get the method optimized, adapted, and ready to work, you have a protocol that can be repeated multiple times in a rigorous way.
Q: Can you explain more about the user friendliness of the chromatography software and how that has helped?
A: Chromatography software in the past was very painful to use, but now the software has become very intuitive and user friendly. It’s not perfect, but I am hoping that in the next couple of years it will improve even more. In our case, the vendor, Bio-Rad (Hercules, CA), provided training on-site for two or three days and helped us understand the instrument and software. It also depends on your knowledge level. After 20 years of chromatography experience, as in our case, the training becomes more specific to the instrument and software and less about understanding the technique. For us, Bio-Rad provided good hands-on training and went over the entire purification procedure to give [us] the confidence that it works. [The trainers] are also quick to respond to any questions we now have regarding the system. One of the advantages of the Bio-Rad Chrom- Lab™ software is that it’s freely available to people in academia, and we can install it on as many computers as we want and do all the modifications to the protocols off-line. All our computers are connected to a shared cloud environment, so you can change and optimize the methods using the software on any computer and put it back into the system and continue to modify it. You can also work on the software from a remote location. You can access the data on a phone or tablet, but you need a large screen to scroll down to look at the various steps listed in the method and to view the details in the different chromatograms. There’s a lot of information to look at.
Q: Have you been successful in coupling and integrating these chromatography systems with other systems?
A: Yes, but mostly off-line. What we are doing with these low-pressure chromatography systems is to get pure protein and do biophysical analysis, either online or off-line. The online detection uses [ultraviolet light] and conductivity. The off-line detection includes fluorescence and nuclear magnetic resonance to look at structural properties and protein interactions. With the chromatography systems that we have, you can perform biophysical characterizations, such as using size exclusion chromatography to look at protein interactions that correspond to the size changes of the proteins.
Q: Are there many challenges with sample preparation for this application?
A: When you perform soluble-protein purification, you start with a cell lysate, where you lyse your cells, mostly mechanically, and spin [them] down in a highspeed centrifuge. The pellet is discarded and the soluble proteins that are in the cell lysate are filtered and then loaded onto a purification column on the chromatography system. We can automate the loading process using sample pumps and load up to half a liter at 1-2 mL per minute in a robust manner. If you do it overnight, you can save a lot of time. Or we can start out with a protein that is semi-purified and load it onto the column to refine it further, to go from 80 to 90 percent purity to 95 to 98 percent purity. There is a lot of flexibility in how you load the samples onto the column using sample pumps or loops.
Q: Are there checks and balances in the system to prevent errors?
A: The system can’t sense [when something] goes wrong, but it does guide you through the setup and protocols to make sure there are no errors. This is a new and important feature in chromatography systems, where it guides the user visually to make sure things are done right the first time. We have our chromatography system running on an uninterrupted power supply, so we don’t worry about power outages. These systems are expensive, but if you need pure proteins, then you must have one.
Wolfgang Peti is a professor in the Department of Chemistry and Biochemistry at the University of Arizona, the Homer C. and Emily Davis Weed Endowed Chair in Chemistry, and an American Diabetes Association Pathway to the Cure Fellow. In addition, he is an associate editor of the Journal of Biological Chemistry, a permanent member of the American Diabetes Association’s Research Grant Review Committee, and a permanent member of the National Institutes of Health/Molecular and Integrative Signal Transduction study section. Dr. Peti received his PhD in chemistry from the University of Frankfurt, Germany, in 2001. He extended his research into structural biology at The Scripps Research Institute from 2001 to 2004 and then at Brown University from 2004 to 2016, where he rose through the ranks from assistant professor to professor. His current research efforts focus on enzyme function related to cancer, diabetes, and neurobiology as well as drug design. Dr. Peti is particularly interested in combining different molecular techniques to understand the function of kinases and phosphatases.