What is an isotope? Isotopes and the mass spectrum

An isotope is a form of an element whose atoms have the same number of protons but a different number of neutrons, so they have different masses. Carbon-12 and carbon-13 are isotopes of carbon: both have six protons, but carbon-13 carries one extra neutron. Isotopes behave almost identically in chemical reactions, so they are easy to ignore in everyday chemistry. In mass spectrometry they can't be ignored: they are the reason a pure peptide gives a cluster of peaks rather than one. This guide explains what isotopes are, how they are written, how common each is, and how to read the isotope pattern of a peptide.

Terms are defined in the glossary.

What is an isotope?

An atom has a small, dense nucleus made of protons and neutrons, with electrons around it. The number of protons, the atomic number, decides which element it is. Every carbon atom has 6 protons, every nitrogen atom 7.

The number of neutrons can vary. Atoms of the same element with different numbers of neutrons are isotopes of that element. They have:

• the same number of protons and electrons,
• almost the same chemistry, because chemistry depends on electrons, and
• different masses, because each neutron adds about one dalton.

The mass number is protons plus neutrons. Carbon-12 has 6 protons and 6 neutrons. Carbon-13 has 6 protons and 7 neutrons.

How isotopes are written

An isotope is written with its mass number as a superscript before the element symbol, or with the mass number after the name (OpenStax Chemistry 2e):

Hydrogen-2 is the one isotope with its own everyday name, deuterium, and its own symbol, D.

How common is each isotope?

Most elements in peptides have one main isotope and one or two rare heavier ones. These are the representative natural shares measured by NIST:

The shares are not perfectly fixed. They vary a little between natural sources, which is why the Commission on Isotopic Abundances and Atomic Weights now gives some atomic weights as a range (CIAAW).

How to calculate relative atomic mass from isotopes

The relative atomic mass on a periodic table is the average mass of an element's atoms, weighted by how common each isotope is. To work it out, multiply each isotope's mass by its share, as a decimal, and add the results.

Worked example: chlorine. Natural chlorine is about 75.76% chlorine-35 and 24.24% chlorine-37 (NIST).

The result, about 35.45, sits inside the standard atomic weight range NIST gives for chlorine, 35.446 to 35.457. The school version, using the mass numbers 35 and 37 instead of the exact masses, gives 0.7576 × 35 + 0.2424 × 37 ≈ 35.5.

The same method gives carbon's 12.011 from carbon-12 and carbon-13. For molecules, the molecular weight calculator adds up the atomic weights for a whole formula, and the sequence properties tool does the same for a peptide sequence.

Stable and radioactive isotopes

Most isotopes of the elements in peptides are stable: their nuclei don't change over time. Carbon-13, nitrogen-15 and deuterium are all stable.

Some isotopes are radioactive. Their nuclei break down over time and give off radiation. Carbon-14 is the familiar example, present in nature only in traces. Radioactive isotopes play no part in the measurements in this guide. Everything below concerns stable isotopes.

Average mass and monoisotopic mass

Because elements are mixtures of isotopes, a molecule's mass can be given two ways: the average mass, from the standard atomic weights, which is the molecular weight used for weighing, and the monoisotopic mass, from only the lightest common isotope of each element (¹²C, ¹H, ¹⁴N, ¹⁶O), which is the first peak in a mass spectrum's cluster. The molar mass, molecular weight and monoisotopic mass guide explains how each is calculated.

Isotopes in a peptide's mass spectrum

A mass spectrometer sorts molecules by mass. A sample of one pure peptide contains molecules with no heavy isotopes, molecules with one, molecules with two, and so on. Each of these has a slightly different mass, about 1 Da apart. So a pure peptide shows as a cluster of peaks, called the isotope pattern or isotope envelope.

A worked example: BPC-157

BPC-157 has the formula C₆₂H₉₈N₁₆O₂₂ and a monoisotopic mass of 1418.704 Da (PubChem). With 62 carbon atoms, each with a 1.07% chance of being carbon-13, many molecules contain at least one.

The heights of the first few peaks, relative to the monoisotopic peak, can be estimated from the abundances in the table above:

The "carbon-13 only" column is a simple binomial estimate. The "all elements" column also counts nitrogen-15, deuterium and the heavy oxygen isotopes. Both are approximate: real spectra differ with the instrument, the charge state and noise.

Two things follow:

• The tallest peak isn't always the monoisotopic one. For peptides above roughly 1,800–2,000 Da, the M+1 peak becomes taller than M. In larger peptides and proteins, the monoisotopic peak can be hard to see at all.
• The pattern checks the formula. A cluster whose shape doesn't match the formula suggests an overlapping impurity or a mistaken assignment.

When the peptide carries more than one charge, the peaks sit closer together on the m/z axis: 0.5 apart for a 2+ ion, 0.33 apart for a 3+ ion. Reading that spacing is one way to tell the charge state. See LC-MS, electrospray and MALDI.

Isotopically labelled peptides as standards

A peptide can be made with heavy isotopes built in on purpose, for example with one amino acid containing carbon-13 and nitrogen-15 in place of the lighter forms. The labelled peptide behaves the same way chemically as the unlabelled one. It comes off an HPLC column at the same time and ionises in the same way. Its mass is a few daltons higher, so the mass spectrometer sees it separately.

That makes it a near-perfect internal standard. A known amount of the heavy peptide is added to a sample, and the ratio of light to heavy signal gives the amount of the unlabelled peptide. The approach is widely used for absolute quantification by mass spectrometry (Gerber et al., PNAS 2003). It is an analytical technique: the labelled peptide is a measuring tool in the instrument, nothing more.

Related guides

For how mass spectrometry confirms a peptide's identity, see mass spectrometry and identity confirmation. For the arithmetic of average and monoisotopic mass, see molar mass, molecular weight and monoisotopic mass. For charged atoms rather than heavier ones, see ions: cations, anions and charge. An independent lab tests every lot we sell for purity by HPLC and identity by mass spectrometry. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.