HPLC shows how much of a sample is one component. It does not show what that component is. For that, peptide analysis relies on mass spectrometry, which measures the mass of the molecules in a sample precisely enough to tell whether the main component is the intended sequence. This article explains how the measurement works, how to work out the mass a peptide should have, and how to read the result.
Principle
A mass spectrometer does three things. It turns molecules into gas-phase ions, separates those ions by their mass-to-charge ratio (m/z), and counts them. The output, a mass spectrum, plots signal against m/z. Peptides are fragile, so they are ionised by "soft" methods that add or remove protons without breaking the molecule apart. Two are standard.
Electrospray and MALDI
| Electrospray ionisation (ESI) | Matrix-assisted laser desorption/ionisation (MALDI) | |
|---|---|---|
| How ions form | A solution is sprayed through a charged needle; droplets evaporate and leave protonated ions | The sample is mixed with a light-absorbing matrix, dried, and struck with a laser pulse |
| Typical ions | Several charge states, [M+H]+, [M+2H]2+, [M+3H]3+ and more for larger peptides | Mostly singly charged, [M+H]+ |
| Usual analyser | Quadrupole, ion trap, time-of-flight or Orbitrap, often coupled directly to HPLC (LC-MS) | Time-of-flight (MALDI-TOF) |
| Strengths | Couples to chromatography, so each HPLC peak can be identified | Fast, simple spectra, tolerant of salts |
Both were developed in the late 1980s; John Fenn and Koichi Tanaka shared part of the 2002 Nobel Prize in Chemistry for the soft ionisation of biological macromolecules.
Working out the expected mass
The mass a peptide should have is calculated from its elemental formula. For mass spectrometry the relevant figure is usually the monoisotopic mass, which uses the most abundant isotope of each element. It differs from the average molecular weight used for weighing, which reflects the natural mix of isotopes.
Modifications change the mass by fixed amounts, which is why they matter for identity:
| Modification | Change in mass (Da) |
|---|---|
| C-terminal amide instead of free acid | −0.984 |
| N-terminal acetylation | +42.011 |
| Disulfide bond (from two free cysteines) | −2.016 |
| Methionine oxidation | +15.995 |
| Deamidation of asparagine or glutamine | +0.984 |
| Residual tert-butyl protecting group | +56.063 |
| Residual trityl protecting group | +242.110 |
| Residual Pbf protecting group (arginine) | +252.082 |
Two of these are worth a second look. Amidation and deamidation move the mass by almost the same amount in opposite directions, and both are close to the 1 Da spacing between isotope peaks, so they need a well-resolved spectrum to tell apart.
Reading a spectrum
Each ion appears at m/z = (M + z × 1.00728) ÷ z, where M is the monoisotopic mass, z the number of protons gained and 1.00728 the mass of a proton. Using triglycine (Gly-Gly-Gly, C6H11N3O4, monoisotopic mass 189.075 Da) as a worked example:
| Ion | Expected m/z | Notes |
|---|---|---|
| [M+H]+ | 190.082 | The usual main ion in MALDI and for small peptides in ESI |
| [M+2H]2+ | 95.545 | Larger peptides carry more charges; the series converges on the same M |
| [M+Na]+ | 212.064 | A sodium adduct, 21.98 above [M+H]+; common and harmless |
| [M+K]+ | 228.038 | A potassium adduct, 37.96 above [M+H]+ |
Some practical points when reading a spectrum:
- Isotope spacing reveals the charge. The peaks within one isotope cluster are about 1 ÷ z apart: 1.0 for a singly charged ion, 0.5 for a doubly charged one.
- Adducts are not impurities. Sodium and potassium ions picked up from glass and solvents give peaks a fixed distance above the protonated ion.
- Agreement is judged against the instrument. A high-resolution instrument agrees with the calculated mass to a few parts per million; a routine MALDI-TOF measurement is typically reported to one decimal place.
- Peaks at other masses can be informative. A peak 16 Da above the main ion suggests oxidation; one a residue's mass below suggests a deletion sequence; one at roughly twice the mass suggests a dimer.
Confirming the sequence
An intact mass confirms the composition, not the order of the residues. Two peptides with the same amino acids in a different order have the same mass. Tandem mass spectrometry (MS/MS) addresses this: a selected ion is broken apart, usually at its peptide bonds, and the masses of the fragments are measured. Fragments that keep the N-terminus are called b ions and those that keep the C-terminus y ions. Ladders of b and y ions, each step one residue apart, read out the sequence.
What mass spectrometry cannot tell you
- Mirror images. A peptide and its D-amino-acid epimer have identical masses. Separating them needs chromatography.
- Quantity. Ion signals depend on how readily each molecule ionises, so peak heights are not a reliable measure of how much is present. Purity comes from HPLC; content from separate methods.
- Isobaric residues. Leucine and isoleucine have the same mass and are not distinguished by routine methods.
- Anything that does not ionise well. Some impurities are under-represented or invisible in the spectrum.
Mass spectrometry on a certificate of analysis
On a certificate, the identity result should name the technique (for example ESI-MS or MALDI-MS) and show the spectrum or the observed mass, which should agree with the mass calculated from the stated sequence and modifications. The article on reading a certificate of analysis covers the rest of the document.
Frequently asked questions
- Why is the observed mass about 1 Da higher than the molecular weight?
- Usually because the spectrum shows [M+H]+, the molecule plus a proton, and because it reports the monoisotopic mass while product data often give the average molecular weight. Both differences have to be accounted for.
- Does a correct mass prove the peptide is pure?
- No. It shows that the intended molecule is present. How much of the sample it makes up is measured by HPLC.
- Is MALDI less reliable than ESI?
- No; they suit different purposes. MALDI gives simple, mostly singly charged spectra and is well suited to confirming the intact mass. ESI couples to HPLC and gives more information about individual impurity peaks.
- Why do some spectra show several peaks for one peptide?
- Different charge states, isotope peaks and salt adducts all produce separate signals from the same molecule. They are expected, and each should correspond to the same underlying mass.
Sources & Further Reading
- Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989;246(4926):64–71. doi:10.1126/science.2675315
- Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry 1988;60(20):2299–2301. doi:10.1021/ac00171a028
- Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides [letter]. Biomedical Mass Spectrometry 1984;11(11):601. doi:10.1002/bms.1200111109
- Steen H, Mann M. The abc's (and xyz's) of peptide sequencing. Nature Reviews Molecular Cell Biology 2004;5(9):699–711. doi:10.1038/nrm1468
- D'Hondt M, Bracke N, Taevernier L, Gevaert B, Verbeke F, Wynendaele E, et al. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis 2014;101:2–30. doi:10.1016/j.jpba.2014.06.012
See also
- HPLC purity analysis of peptides
- How to read a certificate of analysis
- Mass spectrometry — glossary
- Molecular weight — glossary
- C-terminal amide — glossary
- N-terminal acetylation — glossary
- Disulfide bond — glossary
- D-amino acid — glossary
For in vitro laboratory research only. Not for human or veterinary use. This library describes chemistry, analytical methods and published laboratory research; it gives no guidance on use in people or animals.