Understanding Mass Spectrometry

Mass spectrometry (MS) measures the mass of molecules. On a peptide report it is usually the identity test: it asks whether the sample contains a molecule with the mass the named compound should have. This guide explains how that measurement works and how far it goes.

What a mass spectrometer measures

A mass spectrometer turns molecules into ions, separates the ions by their mass-to-charge ratio (m/z), and counts them. The output, a mass spectrum, plots ion abundance against m/z. The instrument does not measure mass directly, only m/z, so the charge on each ion has to be worked out to arrive at a mass.

LC-MS

In liquid chromatography–mass spectrometry (LC-MS), the sample is first separated by liquid chromatography, as described in Understanding HPLC Results, and the column outlet feeds the mass spectrometer. Each chromatographic peak can then be given a mass, which ties the separation and the identity measurement to the same components.

How peptides are ionized

Peptides are most often ionized by electrospray ionization (ESI), which moves molecules from solution into the gas phase with little fragmentation. ESI commonly adds one or more protons, so a single peptide can appear as several ions, written [M+H]+, [M+2H]2+, [M+3H]3+ and so on, each at a different m/z. Software can combine this series of charge states into one neutral mass, a step called deconvolution.

Monoisotopic and average mass

Most elements occur as more than one isotope. Carbon, for example, is mostly carbon-12 with about one percent carbon-13, so a peptide appears not as one mass but as a cluster of isotopic peaks.

• Monoisotopic mass is the mass of the molecule built only from each element's most abundant isotope. High-resolution instruments separate the isotopic peaks and can report this value.
• Average mass, the molecular weight, weights each isotope by its natural abundance. It is the figure registries list as molecular weight.

For a peptide of moderate size the two can differ by a dalton or more, so a report should make clear which one it compares.

Expected and observed mass

An identity test compares the observed mass with the expected mass calculated from the molecular formula. Mass accuracy is often expressed in parts per million (ppm): the difference between observed and expected mass, divided by the expected mass, multiplied by one million. The acceptance limit is set by the method and depends on the instrument's resolution.

What a mass match shows, and what it does not

A matching mass is strong evidence that a molecule of that elemental composition is present. It is not proof of structure.

• Isomers, molecules with the same formula arranged differently, have the same mass. A peptide with two amino acids swapped in its sequence, or with a D- amino acid in place of an L- one, can match exactly.
• Presence is not proportion. Signal intensity depends on how readily each compound ionizes, so the size of a signal is not a direct measure of amount.
• A compound that ionizes poorly, or that falls outside the scanned m/z range, can go unseen. Absence from a spectrum is not proof of absence from a sample.

Tandem mass spectrometry (MS/MS), which fragments a selected ion and measures the pieces, can add sequence information, but it is a further analysis, not part of a simple mass match.

Mass spectrometry and purity

MS answers an identity question. The purity figure on a peptide report usually comes from HPLC with UV detection, and the two measurements complement each other; see Purity & Third-Party Testing, Explained. The registry values for formula and molecular weight shown on compound pages are explained in Reading Peptide Identity Data.

Where this site fits

Where a batch report is published, its identity section can be read in full in the COA Library. How to Read a Certificate of Analysis explains how identity is presented on the reports this site publishes.

Sources and references

• USP General Chapter <736>, Mass Spectrometry. United States Pharmacopeia.
• USP General Chapter <1736>, Applications of Mass Spectrometry. United States Pharmacopeia.
• ICH Q2(R2), Validation of Analytical Procedures, International Council for Harmonisation, 2023, on specificity in identification tests.
• E. de Hoffmann and V. Stroobant, Mass Spectrometry: Principles and Applications, 3rd edition. Wiley, 2007.
• J. H. Gross, Mass Spectrometry: A Textbook, 3rd edition. Springer, 2017.

Research use

All materials on this site are supplied for laboratory research use only. They are not for human or veterinary use, and this guide gives no instructions for preparing or using them.