These posts, tagged “Primer,” are posted for two reasons: 1). to help me get better at teaching non-scientists about science-related topics; and 2). to help non-scientists learn more about things they otherwise would not. So, while I realize most people won’t read these, I’m going to write them anyway, partially for my own benefit, but mostly for yours.
My postdoctoral fellowship here at the University of Iowa still involves research on the mechanisms by which Parkinson’s disease progresses, much like my research at Saint Louis University, but I’m employing different techniques. In an effort to explain those techniques, I’m going to try outlining them here, as it’s a technique that’s “tossed around” on shows like “CSI:” on an almost weekly basis.
Mass Spectrometry is a technology developed over 100 years ago and has been employed by researchers for much of that time. The high cost of procuring one of these instruments (easily in the $10,000s, if not approaching $100,000+) makes them somewhat difficult to find in the undergraduate setting, and sometimes difficult to find in graduate schools. Larger institutions, such as the University of Iowa, will have a few of them, but more than likely, you’ll have to share the instrument with quite a few others, not-so-patiently waiting their turn.
The instrument I’m using is called an LCMS-IT-TOF, pictured above. The acronym stands for “liquid chromatography mass spectrometer – ion trap – time of flight.” Each section of the acronym represents a distinct component of the mass spectrometer: there are different components that can be inserted to achieve similar analytical results in a different fashion. Some components are better for some types of analyses, while other components are better for others.
But, in keeping this relatively simple, I won’t go into it each part. Feel free to check out the Wikipedia article on the subject if you really want to know more about it, but basically, a mass spectrometer is divided into three primary components:
- A source
- A mass analyzer
- A detector
The “source” effectively destroys whatever you’re wanting to look at. There are a variety of different sources one can have in their configuration (e.g. MALDI, ESI, ACPI, etc.) In my case, let’s say you have a protein you want to investigate. The mass analyzer can look at it, but the nature of the type of data it provides makes it much easier to break the protein up into smaller bits first. Therefore, the source breaks up your relatively large molecule of interest (such as the protein in our example) into smaller, more manageable pieces. As with many other things, taking things in “baby steps” is much easier to deal with.
The “mass analyzer” is necessary to help with sorting of all those small, manageable pieces. Think of this process like a box of cereal (I know, right?). Specifically, Frosted Mini-Wheats. When you open the box, you’ll notice that there are mostly fully-formed Mini-Wheats at the top of the box. As you continue on toward the bottom, you’ll start seeing some smaller pieces, some that may have split in half, for example. And at the bottom of the box, you’ll see all the individual wheat fibers and sugar frosting. The same premise holds for a mass analyzer. All those pieces of protein broken up by the source are in different sizes, and the mass analyzer helps sort them out in such a way that the small pieces, medium pieces, and the large pieces are all separated. As with the source, there are many different types of mass analyzers (e.g. TOF, IT, Quadrupole, etc.) used to carry out this work, depending on what you’re looking at.
The “detector” is the piece that really gives us the information we want. After those bits of sample are sorted, they each hit the detector one at a time and the detector tells us what the mass is, typically by actually reading the electrical charge of the sample. Typically, the source (sometimes referred to as an “ionization source”) introduces a charge to each piece of the sample, allowing for the detector to…um…detect them. 🙂
So, how is my work fitting into this? Our lab is interested in how a particular molecule, 3,4-dihydroxyphenylaldehyde (DOPAL) may be involved in Parkinson’s disease. DOPAL is a metabolite of dopamine, the neurotransmitter necessary in order for you to make voluntary movements. When you run out of dopamine (or the cells that produce it, in the region of the brain where you need it), you get Parkinson’s disease. Dopamine is present in those cells, which therefore means DOPAL is present, too. DOPAL is an aldehyde, which means, on a chemical level, it can bind with other molecules relatively easily. What we want to know is whether DOPAL may bind to proteins within those cells. This may matter because cells tend to function in certain ways, and if their individual parts (e.g. DNA, organelles, proteins, etc.) get modified somehow, they won’t work properly and, subsequently, the cell will kill itself to prevent further damage to surrounding cells and tissues.
We want to see whether DOPAL binds to any proteins. If we can find proteins that DOPAL binds to, and if we know what those proteins do inside a cell, then we may be able to a). protect them against DOPAL’s binding, or b). develop drug targets toward those proteins to help prevent them from causing death of the cell.
How does mass spectrometry fit into this equation? Back to our early example of a protein being introduced into a mass spectrometer. The instrument will tell us how much a protein weighs on a molecular level. We also know how much a single molecule of DOPAL weighs. We can, thus, use a mass spectrometer to see whether the mass of a protein increases when DOPAL is present. If that occurs, we can show that DOPAL has bound to the protein. We can also get information as to where on the protein DOPAL bound, or how much DOPAL bound to the protein, and so on.
In the image above (upper left), you can see some vertical lines we refer to as “peaks.” Each peak represents a single mass of a given protein or molecule. You can then take that peak and “fragment” it into smaller peaks. You can do this multiple times (e.g. MS, MS2, MS3 and so on…). Fragmentation patterns give you an idea as to what makes up a complex molecule. For example, if you went from MS to MS2 and had a loss of 18, you could say that you lost a water molecule during fragmentation (O=16, H=1…H2O=18). In the case of DOPAL, we would see an increase in mass (and a shift of the peak) of 151, depending on how DOPAL bound to our protein of interest.
So, basically, that’s what I’m doing in the lab. There’s quite a bit more to the story than this, but I think I’ve simplified the concepts to a mostly understandable level.
Probably not, though. 🙂
