HPLC tells you how pure a sample is. Mass spectrometry (MS) tells you whether it's the right molecule. Here's how to make sense of the numbers on an MS report.
The basic principle
A mass spectrometer turns molecules into gas-phase ions, separates them by mass-to-charge ratio (m/z), and counts them. The result is a spectrum: m/z on the x-axis and signal intensity on the y-axis.
Getting peptides into the gas phase
Electrospray ionisation (ESI)
The sample is sprayed from a charged needle, usually straight from an HPLC column (LC-MS). ESI tends to add several protons, so a peptide shows up at several charge states.
MALDI
In matrix-assisted laser desorption/ionisation, the sample is mixed with a light-absorbing matrix and hit with a laser. MALDI mostly produces singly charged ions, which makes spectra simpler. It's often paired with a time-of-flight (TOF) analyser.
John Fenn (ESI) and Koichi Tanaka (soft laser desorption) shared the 2002 Nobel Prize in Chemistry for making it possible to analyse large biomolecules this way.
Working out charge states
With n protons added, a peptide of mass M appears at m/z = (M + n × 1.007) ÷ n. For a peptide of mass 1500.0 Da:
| Ion | Charge | Observed m/z |
|---|---|---|
| [M+H]⁺ | 1+ | ≈ 1501.0 |
| [M+2H]²⁺ | 2+ | ≈ 751.0 |
| [M+3H]³⁺ | 3+ | ≈ 501.0 |
Software "deconvolutes" these series back into a single neutral mass. That's the figure usually compared with the expected mass on a report.
Monoisotopic versus average mass
Natural carbon is about 1.1% carbon-13, so a peptide appears as a cluster of peaks about 1 Da apart (divided by the charge). The monoisotopic mass uses only the most common isotopes and is the first peak in the cluster. The average mass is weighted across all of them. For larger peptides the monoisotopic peak becomes small, and the tallest peak in the cluster is a heavier isotope. Reports should state which mass they're quoting.
Adducts and common shifts
| Observed difference | Likely cause |
|---|---|
| +22 Da vs [M+H]⁺ | Sodium adduct [M+Na]⁺, common and usually harmless |
| +38 Da vs [M+H]⁺ | Potassium adduct [M+K]⁺ |
| +16 Da | Oxidation, often methionine |
| +1 Da | Deamidation, or simply the next isotope peak |
| −18 Da | Loss of water |
| Minus one residue's mass | Deletion sequence from synthesis |
Confirming the sequence: MS/MS
In tandem mass spectrometry, a selected ion is broken apart, usually along the backbone. The fragments form ladders, called b-ions (from the N-terminus) and y-ions (from the C-terminus). The mass gaps between neighbouring fragments identify each amino acid, so the sequence can be read directly. This can tell apart two peptides with the same overall mass but different sequences, which a single mass measurement can't.
Accuracy matters
High-resolution instruments such as Orbitrap and modern TOF analysers measure mass to within a few parts per million. Lower-resolution instruments may only agree to within ±1 Da. A good report states the instrument and its tolerance.
Related articles
- How laboratories validate analytical methods
- D-amino acids and peptidomimetics
- Why some peptides are hard to make
Sources and further reading
- Fenn JB et al. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989;246:64–71. doi:10.1126/science.2675315 · PMID: 2675315
- Steen H, Mann M. The ABC’s (and XYZ’s) of peptide sequencing. Nat Rev Mol Cell Biol 2004;5:699–711. doi:10.1038/nrm1468 · PMID: 15340378
- Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature 2003;422:198–207. doi:10.1038/nature01511 · PMID: 12634793
- The Nobel Prize in Chemistry 2002 (Fenn, Tanaka, Wüthrich). www.nobelprize.org/prizes/chemistry/2002/summary