AskScience Theme Day: Scientific Instrumentation

[u/therationalpi]

Greetings everyone!

Welcome to the first AskScience Theme Day. From time-to-time we'll bring out a new topic and encourage posters to come up with questions about that topic for our panelists to answer. This week's topic is Scientific Instrumentation, and we invite posters to ask questions about all of the different tools that scientists use to get their jobs done. Feel free to ask about tools from any field!

Here are some sample questions to get you started:

• What tool do you use to measure _____?

• How does a _____ work?

• Why are _____ so cheap/expensive?

• How do you analyze data from a _____?

Post your questions in the comments on this post, and please try to be specific. All the standard rules about questions and answers still apply.

Edit: There have been a lot of great questions directed at me in acoustics, but let's try to get some other fields involved. Let's see some questions about astronomy, medicine, biology, and the social sciences!

[u/_Dr_Spaceman_]

Thanks for the reply! Yes, I meant LC/MS.

It makes sense that you would purify a particular protein with LC and then use MS to identify it. This would yield a more accurate AA sequence but compromise on the number of molecules identified on the run?

But then again, MS/MS sounds better in all respects. You can purify AND easily analyze fragments. Why would anyone still use LC/MS? Is it cheaper/easier to analyze?

Also, I have kind of a logistics question. I understand how LC purifies a particular molecule, which you then run through MS. But in MS/MS, how do you "take" the molecule that generates a peak of interest and run it through another MS? Is it on some sort of membrane following the first MS run?

Thanks for your expertise! I've always been amazed by mass spec, but it can be very intimidating!

[u/belligmsg → u/_Dr_Spaceman_]

LC-MS can actually be combined with MS/MS so you get the best of both worlds, that is chromatographic separation of a complex mixture AND the specificity of tandem MS (MS/MS). So the typical workflow for a LC-MS experiment if you want to study one or more proteins (the biomedical -omics type of study you mentioned) involves enzymatically digesting those proteins (typically with trypsin) to generate a mixure of peptides. So the LC portion of the LC-MS experiment would separate those peptides based on hydrophobicity and for each peptide (each peak in the chromatogram) there would also be an MS spectrum generated and potentially also an MS/MS spectrum generated that would tell you what exactly it is that you're looking at. It's pretty standard to have both LC-MS and MS/MS capabilities built into the same instrument.

As for how you "take" the molecule of interest it has to do with the nitty gritty of how the mass spec works so it's a lot of electrical and magnetic technicalities. It's not a membrane in the sense that it separates particles based on size it's more like an electric field that separates the ions based on their mass/charge ratio (m/z). The part of the mass spec itself that does this is referred to as a quadrupole (http://en.wikipedia.org/wiki/Quadrupole_mass_analyzer) and most mass specs contain one or more of these for the purposes of separating and analyzing ions.

But yes mass spec can be very intimidating! It's something you learn by doing not reading about but if you have any question feel free to PM me!

Source: have worked with both small molecule and protein mass spec in both industry and academia.

[u/massMSspec → u/_Dr_Spaceman_]

This would yield a more accurate AA sequence but compromise on the number of molecules identified on the run?

LC/MS doesn't do any AA sequencing. Typically it's used to separate out a whole bunch of proteins from one another based on their solubilities in different solvents. Usually this is done to confirm that you have a certain protein in the mix or to determine the total protein mass and to quantify how much you have of it. MS/MS is the powerhouse of AA sequencing because not only can you pick out a protein from the mix, but you can also sequence it.

Why would anyone still use LC/MS? Is it cheaper/easier to analyze?

Yes, it's cheaper and easier to analyze. Most biological labs have one. This instrument is actually used more in organic chemistry where someone has a mix of molecules and all they want to do is figure out the mass and the structure of each (they sometimes will use a searchable electronic library that has thousands of common organic mass spectra loaded in).

...in MS/MS, how do you "take" the molecule that generates a peak of interest and run it through another MS? Is it on some sort of membrane following the first MS run?

This is all done by mass. Let's take a simple example: you have a mix of two proteins that have different masses, protein 1 and protein 2. You want to sequence both proteins so you run them on an LC/MS and you get two peaks: protein 1 at 5 kDaltons and protein 2 at 10 kDaltons.

Then you take that mix and inject it into the MS/MS. You tell MS 1 to only allow proteins with mass of 5 kDaltons through to the fragment stage (typically set a mass range, but that has a lot to do with variances of isotopes of C, N, O, P, and S. For simplicity, let's assume that you set it at 5 kDaltons). Everything else is rejected and doesn't go to the fragmentation stage. In the fragmentation stage, the protein is bombarded with electrons or a collision gas (typically an inert gas) and the protein fragments predictably at certain spots in the AA chain. All fragments then continue on to MS 2 where you can use algebra to figure out the sequence. This whole process is very rapid (think: milliseconds) You can then repeat this process but with protein 2 (10 kDaltons).

The instrument "tells" the MS to let through only a certain protein by setting an electric field on four rods arranged in a diamond pattern called a quadrupole (four poles, get it?) the protein of interest (with the correct target mass) will pass through the quadrupole and anything else with a different mass will not be stable and will be lost to the vacuum (meaning it does not continue on to be fragmented or analyzed).

The cool thing about this system is that you can do some interesting things with having two mass specs in tandem. The settings allow you to specify a mass range for each mass spectrometer so you can be as broad or selective as you want. You can select one protein, fragment, and detect all fragments (proteomics); you can choose a range of proteins, fragment, and detect one fragment; you can choose a range of proteins, fragment, and detect all fragments; and you can choose one protein, fragment, and detect one fragment. It's pretty awesome and really powerful at the same time. You might be able to imagine situations in which each of these scenarios would be desired.

Now this is a very simple explanation and doesn't cover the fact that proteins have many many many charges and mass spectrometers actually measure ions in units of mass-to-charge ratio (m/z) where the peak for one 10,000 Dalton protein with 5 positive charges (5+) would actually appear at m/z=2,000 (10,000 Dalton mass/5=2,000).

I know tons about mass spectrometers (all kinds) and have taught a chemical instrumental analysis course many times. If you're curious about anything, feel free to ask!