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How Does Mass Spectroscopy Work?

Mass Spectrometry, also referred to as mass spec, is an analytical technique becoming increasingly important in bioscience research.  This article will introduce you to this technique, how it works, and how it could be useful to you in your research.  So let’s get started with your introduction to mass spectroscopy…

What Is Mass Spectrometry?

In a nutshell, mass spectrometry accurately measures the mass of different molecules within a sample. Even large biomolecules like proteins are identifiable by mass, which means that biologists can perform some very interesting experiments using mass spectroscopy, potentially adding a new dimension to your research.[1]

Could Mass Spectroscopy Be Useful for Me?

Yes, if you want to:

  • Identify molecules in a mixture
  • Detect impurities in a sample
  • Analyze a purified protein
  • Study the protein content of a sample of cells

How Does Mass Spec Work?

There are three key stages to the spectrometer:

1.  Ionization.

Molecules in a sample are vaporized (converted to the gas phase by heating). Then, an electron beam bombards the vapors, which converts the vapors to ions. Because mass spectroscopy measures the mass of charged particles, only ions will be detected, and neutral molecules will not be seen.

Ions are created by giving electrons to a molecule (producing a negatively charged ion) or taking electrons away from a molecule (producing a positively charged ion).

Note: a sample can only be analyzed by mass spectroscopy if it can be vaporized without decomposing.

2.  Acceleration and Deflection.

Next, the ions are sorted according to mass in two stages – acceleration and deflection.

Acceleration is simple attraction. The positive ions created in the ionization stage accelerate towards negative plates at a speed dependent on their mass. In other words, lighter molecules move quicker than heavier ones.

Deflection is the magnetic bit…the ions are then deflected by a magnetic field, and the extent of deflection is again dependent on mass.  So, ions of different mass travel through the spectrometer at different speeds.

3.  Detection.

Ions of increasing mass eventually reach the detector one after another, and then it’s over to the computer to provide a spectrum. The output will look something like in Figure 1.

Figure 1. Sample spectrum produced by mass spectrometer. Simplified mass spectrum of pentane (CH3CH2CH2CH2CH3).

Note:  This simplified mass spectrum has been drawn using data from the Spectral Data Base System for Organic Compounds (SDBS) at the National Institute of Materials and Chemical Research in Japan.

Omission of minor lines with peak heights of 2% or less of the base peak (the tallest peak) provides clarity.

Reading The Spectrum

First, you should notice that simply ‘mass’ on the x axis but ‘m/z’, where m is the mass and z is the charge. What the spectrometer actually measures is mass/charge (m/z) ratio. Sounds complicated, but it’s not—because the majority of the ions produced during MS have a charge of 1, m/z is normally equal to the mass of the molecule. Simple.

Is Mass Spectroscopy Worth It?

In a word: Yes.  Mass spectroscopy provides accurate weight measurements for your bio- (or other) molecules, which can be used to:

  • Give a good estimate on the purity of the sample (i.e., whether there is one or more molecular species in your sample and what ratio those species are in)
  • Monitor reactions, sequence amino acids, and oligonucleotides as well as give information on protein structure
  • Identify and quantify samples

Mass spectrometry has enabled biology to move from identifying single proteins to proteome-wide characterization and quantification. With the development of this tool, researchers can now feasibly begin defining biochemical pathways on a kinetic basis, which will help us understand the mechanistic response of cells to changing environments.

Peptides, Hospitals, Drugs and Crime Scenes: Practical Uses of Mass Spectroscopy

Drug companies and researchers are using the technique for drug discovery, for gaining information on drug metabolism and for pharmacokinetic studies.The study of proteins through mass spectrometry is now aided by information obtained from the genome sequencing projects.3  The technique has also found medical uses, such as neonatal screening, blood analysis, and drug testing.  These uses also make mass spectroscopy (when coupled with other analytical techniques) a powerful tool in forensic analysis.

How Do I Go About Using Mass Spectroscopy?

If you feel that mass spectroscopy is something that could be of use in your research, check whether your department or college has mass spectrometers available for use. This might be either as a ‘walk up’ service where you book a slot and run your own samples or as a technician-run service. Training will always be provided for these services so ask around.  While the number of uses is large, the instrument itself is not —normally the machine can fit on a bench top.

But Remember This Before You Use a Communal Mass Spectrometer…

The most important factor in getting accurate results is keeping it clean and free of contaminants. As many a chemist will testify, this is often an issue with communal mass spectrometers—especially if the user before you did not the column properly. It’s wise to bring your own solvent to clean the column before (and after!) use.

Want to Know More?

This article was a brief introduction to the basics of mass spectrometry, but it doesn’t end there! There are various ways of producing the ion required, and the method chosen depends on the nature of the sample molecule. You can find out more information in the references below.

References

  1. Baker M. (2010) Mass spectrometry for biologistsNature Methods. 7:157.
  2. Ashcroft AE. An Introduction to Mass Spectrometry.
  3. Yates JR. Mass spectrometry in biology. In: Encyclopedia of Life Sciences. pp. London: Nature Publishing Co.; 2001. 1–5.

Originally published in 2011, republished in 2016.

Image credit: Lachlan Arthur

8 Comments

  1. Matteo on November 10, 2016 at 1:34 pm

    Hello, This article has really been helpful to me for a school project!!
    One thing I do not entirely grasp is how you can tell which molecules are being separated, and which molecules are which at the end. For example this machine can also be used to test athletes to see if they have been taking drugs. How does it do that?
    Thank you again for all this information

  2. Jasper Whiteside on October 27, 2016 at 5:41 pm

    I’m glad the article mentions that a mass spectrometer can be used in forensics because I was trying to remember where I had heard of it before. The answer is from a TV show that I used to watch. It really is fascinating how powerful these machines are in analyzing biological materials.

  3. Allison Ross on February 28, 2011 at 1:53 pm

    Lawrence – that great to know! We’ll be taking the series through the basics first, looking at why this particular technique works in the way that it does. Parts 2 and 3 are coming up shortly which explain some of the different methods of ionizing your sample and compare ESI with MALDI. Later, we’ll be looking at more specific uses and functions in a biological frame. Keep telling us what you want, and we’ll do our best to find the information for you. Thanks!

    • incommunicada on July 21, 2012 at 8:09 am

      Do you have an article on how to read ESI/MALDI spectra? I haven’t fully grasped how yet.

  4. Lawrence Forsberg on February 26, 2011 at 6:13 am

    Tally me as another person who’s really enjoyed the articles lately.

    I’m personally interested in the identification of metabolites in cells under different conditions (drug treatments, knockouts of different genes, etc.) to identify the role of uncharacterized enzymes, and things like post-translational modification mapping.

    Any chance your next article (or another some time soon) will cover any of these?

    Thanks again for the good read!

  5. Carl Saxton on February 23, 2011 at 5:12 pm

    Thanks for your comment. I am glad you enjoyed reading the article. I’d be interested in knowing what part of the article interested you most? Is there any specific Mass spectrometry topic you would like us to cover?

    • veena on October 19, 2012 at 12:41 am

      Hello Mr.Saxton, This article has been extremely helpful to me in understanding how ionization occurs. Thank you very much! However I have various other doubts on how the sample is processed. For instance in HPLC/ESI-MS initially the sample undergoes liquid chromatography. Different compounds gets separated based on their mass and gets eluted at different times based on the retention times. So the elution from the HPLC is discontinuous. Does the MS analyze the sample as and when it gets eluted from HPLC and gives a spectrum for each such event? I hope this question makes sense. I basically want to know what exactly happens to the sample after the ionization process.

  6. Amin on February 22, 2011 at 6:35 pm

    These are really great series. I look forward learning more. Thanks a lot!

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