Mass Spectrometry

Mass spectrometry is now a standard technique for analysing peptides and other chemical compounds. In peptide research it may be useful to determine the molecular mass of a peptide, to check the identity and to investigate the presence of the expected compound in a sample.

The idea is simple. A sample is converted to charged particles, called ions. These ions are separated based on their mass-to-charge ratio (m/z), and the output data allows scientists to know what is in the sample.

This is useful in peptide studies because peptides with similar properties may have different sequences or molecular masses.

What do you measure in mass spec?

Mass spectrometry doesn’t weigh a peptide the way a normal set of scales does. Instead, the device detects ions by their mass-to-charge ratios.

The peptide is introduced into the instrument and ionised. These ions are then sent through a mass analyser which separates the ions according to their m/z values. The ions are then detected and a mass spectrum generated.

The spectrum normally appears as a series of peaks. Each peak represents the ions that exist at a certain m/z value. The height of a peak correlates to the strength of the ion that is detected.

“That gives the researchers a nice chemical fingerprint of the sample.”

What is the role of mass spectrometry in peptide research?

Peptides are chains of amino acids linked in a specific order. Even a slight change in that sequence can change the molecular weight of the peptide.

Mass spectrometry can therefore be used by researchers to confirm that a synthesised peptide has the expected mass.

If the measured value differs from the expected value, the sample might require further investigation. The difference may be due to incomplete synthesis, modification, degradation or presence of another compound.

Mass spectrometry can also be used to identify other molecular species present in the sample. It is generally used alongside other analytical methods and not as a full test in itself.

How does a mass spectrometer function?

A mass spectrometer has several stages, but the specific design of the instrument varies.

The sample is initially introduced and ionised. For peptide analysis the most common are electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI). These techniques allow the analysis of relatively large molecules like peptides.

The ions pass into a mass analyser where they are separated according to their mass to charge ratio.

Finally the ions reach a detector. The instrument records the signals, and the software translates the measurements into a mass spectrum.

There are several different types of mass analyser, including quadrupole, time-of-flight (TOF), ion trap and Orbitrap systems. The type most appropriate depends on the research question and on the level of information required.

What is m/z?

“M/z” is one of the main terms in mass spectrometry, meaning “mass-to-charge ratio.”

This is important because peptides can be charged in multiple ways. This means that the same peptide can produce different m/z values depending on its charge state.

This is why a single peptide may show up as multiple peaks. Researchers use these charge state patterns to determine the underlying molecular mass.

The multiple charging is especially helpful when performing analysis of larger peptides, since it provides the possibility to detect them in the working range of the instrument.

Peptides: Purity and mass spectrometry

Mass spectrometry can be a useful source of information about a peptide sample, but it should not be considered an automatic complete measurement of purity.

In addition to supporting the identity of a target peptide, a mass spectrum needs to be complemented by another method to assess how much of the sample is that peptide.

High performance liquid chromatography (HPLC) is frequently coupled to mass spectrometry. HPLC separates the components in a sample, and mass spectrometry can be used to help determine the mass and identity of compounds corresponding to the peaks detected.

The two techniques thus yield different, but complementary information.

LC-MS is the combination of liquid chromatography and mass spectrometry.

What is tandem mass spectrometry?

For a more detailed analysis of the structure, researchers can use tandem mass spectrometry (MS/MS).

In MS/MS, an individual peptide ion is isolated and broken into smaller fragments. The fragments are then analysed for additional information about the molecule.

This can be useful in peptide research for studying sequences of amino acids. Instead of just asking what the molecular mass is, researchers can see if the fragments match the expected peptide sequence.

This is the rationale for tandem mass spectrometry for peptide identification and sequence analysis.

What can affect the results of mass spectrometry?

Mass spectrometry is powerful. Results are dependent on sample preparation and instrument conditions.

Contamination can give spurious signals. The quality of the data can also be affected by the sample concentration, the solvent conditions and the ionisation method chosen.

Peptides can undergo changes during storage or preparation. Oxidation, hydrolysis and other degradation may result in signals different from those predicted for the parent peptide.

The instrument also requires calibration and proper maintenance. As such, the results must be considered within the framework of the entire analytical method rather than examining a single peak in isolation.

Mass spectrometry in the research of peptides

The use of mass spectrometry in peptide research varies from analysing the molecular mass of a synthesised peptide to degrading products and studying peptide sequences.

Its main value is the molecular information that it provides. It can be used in conjunction with HPLC and other analytical techniques to help researchers better understand what is present in a peptide sample.

The idea is straightforward. Mass spectrometry measures ion parameters in accordance with their ratio of mass to charge. This allows for the obtaining of information that can be used to characterise compounds, as well as analyse them.

Knowledge of this elementary concept becomes useful while interpreting the mass spectra of peptides and putting the results obtained in analysis into the proper context in research.