How Does Mass Spec Work? Part 2 – Ionization

by on 6th of April, 2011 in Analytical Chemistry

In the first part of our “introduction to mass spectrometry” series, we established that mass spec is a very useful technique that biologists can use for a number of applications, from analyzing a purified protein to studying the protein content of a sample of cells.

We also discussed that ions, not molecules, are detected and measured by the mass spectrometer and that those ions must be in the gaseous phase.

So how can you turn your nice sample into a gaseous cloud of ions that can be analysed using mass spec? In this article, we’ll take a look at the various ionization techniques used in mass spec, and see which one you should be using for your biological samples.

How are ions created?

Broadly speaking, ionization methods can be split into two groups: ‘hard’ and ‘soft’ ionization.

• Hard ionization – results in the breaking of chemical bonds and the formation of fragment ions.

• Soft ionization – results in the formation of ions without breaking any chemical bonds. In other words, all covalent interactions are kept intact. In fact, sometimes non-covalent interactions can be kept intact! This really is a big deal – and is covered in much more detail when we look at ESI (Electrospray Ionization) in a later article.

Usually the ions formed in MS are positively charged (cations), although negative ions (anions) can also be generated. Whether you run your MS experiment in positive or negative ion mode is dependent on your sample. For example, DNA is negatively charged, so you’d probably choose to run the experiment in negative ion mode.

As a general rule and a good starting point, if your sample is basic (e.g. a protein with a high pI), analyze it in positive ion mode, and if it is acidic (e.g. DNA or a protein with a low pI), analyze it in negative ion mode.

Hard ionization

In hard ionization techniques, you have to vaporize the analyte first, and then generate the ions from the neutral molecules that have been introduced into the gas phase (i.e. generated from thermally volatile samples). This is normally accompanied by fragmentation (breaking) of the molecule.

We saw an example of hard ionization in Part 1 where we looked at the fragmentation of pentane. The source of energy for this fragmentation can be high energy electrons (Electron Impact ionization), or a chemical source (Chemical ionization).

Hard ionization techniques are routinely used for small, volatile molecules, and the fragmentation patterns are often very useful to scientists in identifying unknown molecules. However, as biologists, it’s likely that you will want to look at large molecules, in which case ‘soft’ ionization techniques are more appropriate, so we will discuss these in more depth.

Interested in large biomolecules? You need soft ionization

As the name suggests, soft ionization techniques are a bit gentler with your molecule, keeping the molecule intact allowing you to observe the M+ ion.**

**Strictly speaking, we should denote the molecular ion as MH+ or [M+H]+ when we’re talking about soft ionization, because the charge on the analyte tends to come from either the addition or loss of protons (H+) in these “soft” techniques, rather than from the gain or loss of electrons (we’ll talk a little bit more about this below…)

Soft ionization techniques are the preferred methods for large, non-volatile, polar macromolecules such as peptides, proteins, lipids, oligonucleotides, polymers, and oligosaccharides. On certain types of mass analyzers, such as time-of-flight (TOF) instruments, you can routinely analyze ions up to 200 kDa in size, and sometimes you can even look at larger ions than this.

You can mix and match different types of ionization techniques with different types of mass analyzers (mass analyzers differ in the way that they measure the m/z ratio) and we’ll take a look at some of these different mass analyzers, such as TOF, quadrupole and FT-ICR mass spectrometers, in future articles.

Soft ionization

Let’s just emphasize again why we want soft-ionization to look at our biomolecules by mass spectrometry. As we’ve already mentioned, biological macromolecules tend to be polar and non-volatile. This means that they will probably be dissolved in aqueous solution, and heating them i.e. to get them into the gas phase, will just cook them rather than vaporize them! Soft-ionization techniques allow us to ionize these chunky, thermally fragile molecules, and get them from aqueous solution into the gas-phase without giving them too much energy and therefore preventing them from falling apart – and so we can measure their mass!

The most common soft ionization methods are Matrix Assisted Laser Desorption Ionization [MALDI], Electrospray Ionization [ESI] and Fast-Atom Bombardment [FAB]. FAB was used less and less after ESI was introduced – a major reason being that ESI can analyze molecules with a much wider molecular weight range. Also, with ESI there is no matrix, so you don’t get interference from matrix in your mass spectrum.

If you’re using mass spec for your experiment, then you already know whether you want to find out about covalent or non-covalent interactions in your sample. This will determine which soft ionization technique such as MALDI or ESI you use. Here’s something to watch out for – you might see soft ionization techniques sometimes referred to as ‘desorption’ techniques, which is a bit misleading as desorption is the release of molecules from a surface or matrix. As we have already said, ESI uses no matrix. So it’s okay to refer to MALDI as a desorption technique (in fact it’s in the name!) but just be aware that this is not an interchangeable term for soft ionization in general.

Both MALDI and ESI will be covered in separate articles coming up next in parts 3 and 4.

The technique you choose will depend on the nature of your sample and the information you want to obtain. Soft ionization techniques generally produce single or multiply protonated ions in the positive ion mode, so [M+nH]+ or [M+nCation]n+. In the negative ion mode we observe deprotonated ions such as [M-nH]n- or [M-nCation]n-.

A real advantage for these soft ionisation methods is that they can be combined with tandem mass spectrometry [MS/MS] to obtain more detailed structural information. We’ll cover this too in later parts, but for now we’ll take a look at:

Fast atom bombardment – it’s FAB! (Or at least, it was…..)

We should briefly mention FAB as it is a relatively soft ionization technique and can be used to look at non-volatile, polar stuff like peptides, but it has mostly been replaced by the modern-day soft ionization techniques MALDI and ESI.

FAB can still be used for peptide sequencing, but nowadays you’d be far more likely to use tandem MS methods. For biological mass spec, we will concentrate on MALDI and ESI ionization techniques only.

So where are we now?

In 2002, the Noble Prize was awarded for the development of MALDI and ESI – the development of these techniques completely revolutionised the field of biological mass spec and in the next instalment we’ll explain how MALDI works. We’ll take a look at the matrix – what exactly is it? – And why choosing the right matrix is one of the most important considerations in desorption ionization.

ESI is just as, if not more important than MALDI. It’s certainly just as heavily used – look out for these in the next couple of articles in this series.

One thought on “How Does Mass Spec Work? Part 2 – Ionization”

  1. Hi Vicki!

    This is very informative! Do you perchance have the Parts 3 and 4? I couldn’t find it anywhere in the site.

    Thank you!

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