Addressing the Challenges of Small Molecule Analysis

From identifying novel drug targets and cancer therapies to improving Chinese medicine standards, small molecule research spans many fields. However, the protocols in place to perform small molecule characterization and identification are often not as good as they should be.

Technology Networks spoke with Andreas Huhmer, Senior Director OMICS, Life Science Mass Spectrometry at Thermo Fisher Scientific to learn how the new Thermo Scientific Orbitrap IQ-X Tribrid mass spectrometer helps address the complexities of small molecule identification and characterization.

Ash Board (AB): What are the key features of the Orbitrap IQ-X Tribrid that make it suited towards small molecule research?

Andreas Huhmer (AH): The Orbitrap IQ-X Tribrid mass spectrometer is an evolution of the Thermo Scientific Orbitrap ID-X Tribrid mass spectrometer that launched a few years ago and improves upon the themes that we started with the ID-X. Small molecules are chemically very diverse and that means you have to be able to address those molecules in various ways in terms of how they are analyzed. The new instrument has the ability to fragment the small molecules using different energies with different approaches, and this has been improved by adding what we call a mild trapping function. So, if there are labile molecules that tend to fall apart in the process of analysis, they are now preserved so that you can get more structural information about more of these really fragile molecules.

Another feature that we added to the IQ-X addresses chemical diversity. There are a lot of small molecules that have aromatic structures or double bonds, particularly lipids. These can be addressed with a different fragmentation technique, in this case using an ultraviolet photodissociation (UVPD) laser. This puts energy into the molecule so that it falls apart in a distinct way and gives the users an additional ability to look at structurally diverse molecules.

The other way we've improved the IQ-X is its ability to look at very complex mixtures. When we introduced the instrument, we launched something called the Thermo Scientific AcquireX Intelligent Data Acquisition Workflow. With this workflow you start out by providing the instrument with some information that is either focused on the sample or has nothing to do with the sample. A simple example is the introduction of solvent as your background. The instrument stores that information so that when the sample is injected, the instrument simply ignores all of the stuff that does not have anything to do with the sample. Basically, we have improved that intelligence with what we call Advanced Deep Scan Workflow. Here, you can combine the information from various sample sets. This makes it more efficient at finding pieces of structural information that may be important. There are also a number of other improvements that you would expect from a next generation instrument. These include, enhanced low mass range, a segmented quadrupole for better mass filtering and advanced peak determination features.

AB: Another feature you added was real time library search, can you tell me more about this?

AH: If you look at a typical drug discovery effort, where you are trying to understand a drug candidate, you may be investigating a compound with a particular chemical structure, that, when introduced into a mouse or a human is metabolized and typically transformed into other chemical compounds. In a traditional approach it takes a lot of effort to figure out what might happen, so people hypothesize and look for that expected mass. With the IQ-X this is all being done, essentially, in real time. So what you do is you say, here is the molecule of interest, the drug molecule, for example and then please look at any fragmentation spectrum that you acquire and see what relates back to this particular drug molecule. Imagine that the drug molecule falls apart into three pieces; they have particular molecular characteristics at a particular m/z. Every time these show up in a fragmentation event, you say, give me more information, trigger MS3 or spend more time on this peak. This is a very elegant way to find out more about those unknowns that might be in the sample that relate to the drug candidate. Having the instrument do all of the hard work while actually acquiring data is really a breakthrough.

AB: That must add up to significant time savings for the researchers. Do you have any examples of the time savings people have managed to achieve?

AH: Yes, absolutely. In one particular example, the team were looking at a typical drug discovery scenario similar to the one I described above. In a traditional way, for example with the ID-X, they were able to find 11 metabolites. With the IQ-X and its real time library searching capability, they were able to increase this to 17 metabolites. That's a very good example of how this capability can be utilized to make progress in a much more relevant way.

Another good class of analytes to think about is natural products, which are often drug molecules but also lipids. This is an area where we think there will be a lot of excitement. To give another example, drug molecules are typically glucuronidated as part of human metabolism because this is a way for drugs to be excreted in the stool. This adds a double bond, so you can now use the UVPD source. Therefore, if you find the drug molecule, or a part portion of a drug molecule, you can look to see whether there is a glucuronidated form of it. You can quickly add the mass of a glucuronidation group, and then look for that particular mass within your analysis. That's a huge time saver and makes things much more efficient.

AB: What do the AcquireX and the Met-IQ workflows provide labs? 

AH: Imagine a scenario where you treated three groups of rats or mice with a drug. In this particular case, you would like to understand whether these different types of mice react differently to the drug, or if they have a different way to metabolize the drug. Often in research right now, you have transgenic mice, so they have particular phenotypes. Typically, you would run these as three independent experiments and would then try to make sense of the data during analysis. With the advanced deep scan, you can combine information from these three groups of mice and then submit all of this into the deep scan. So deep scan can consider the particular differences originating from each animal group while analyzing them at the same time. For example, you could run the first and second groups of mice and then combine these two pieces of information before you analyze the third group.

AB: According to Thermo Fisher Scientific’s website AcquireX enables unbiased interpretation. Can you explain what is meant by unbiased interpretation and how AcquireX helps researchers achieve this? 

AH: What we mean by unbiased interpretation is the ability to extract more information from the sample. Imagine there are 100 metabolites in a particular sample, but you're only able to identify 30 or 50 of them through your efforts; you're basically ignoring the other half of the information about this drug. You automatically bias your conclusions. With the ability to dig much deeper, you get much less of a bias in your results.

AB: The IQ-X has a new auto-ready ion source, which enables walkway calibration. Can you run me through this and the benefits it provides?

AH: This is probably one of the more exciting features for efficiency. Typically, customers would take time to take the source off and put on another particular source to calibrate the system every few days, because they want to be able to do ppm type accuracies for their small molecule analysis. The better the mass accuracy you have, the more precise your interpretation of data will be for small molecules. In the IQ-X, the source actually auto calibrates the instrument. This isn’t done in a random fashion. The IQ-X can recognize when nobody is using it, for example because the previous run just finished and the next run isn't scheduled, and will use this time to check on the calibration. This doesn't mean it will always require calibration; it may only need a refresh of the calibration. It is automatically set up to do that. Therefore, when the instrument is needed again, it is freshly calibrated and ready to go. It is possible to schedule calibration in many different ways between instruments in different conditions. This is another very good example of how this instrument drives efficiency for the users. And the more accurate your measurements are through strong calibration, the better results you will achieve.

AB: The IQ-X contains a UVPD source, could you just talk to me a little bit about that and what that provides?

AH: The ability to excite molecules through a UVPD laser enables you to fragment and ultimately characterize small molecules that have a double or triple bond. A good example would be lipids which have double bonds in their fatty acids. If you expose them to UVPD they fragment in very characteristic ways. Lipids are particularly challenging because if they don't have a double bond, they don't fragment easily. Using non-chemical terms, the ability to essentially cut the fatty acid group in half with the UVPD laser and then look at the pieces allows you to look at a triglyceride or diglycerides and understand which fatty acids are part of the triglyceride group. UVPD provides a much better way to assess the length of these fatty acid groups which is very helpful, because there's really no other way to do this otherwise.

Andreas Huhmer was speaking to Ash Board, Editorial Director for Technology Networks.