ESI vs MALDI: Mass Spectrometry for Peptide Identity
HPLC says how much of one thing is present. Mass spectrometry says what it is — and on a long peptide that is the more important question.
HPLC tells you how much of one thing is in the vial. It does not tell you what that thing is. Mass spectrometry answers the second question, and on any peptide longer than a few residues it is the more important of the two.
Why identity is a separate question
A chromatogram shows peaks separated by hydrophobicity. A peptide missing one residue is chemically very similar to the full-length product and can elute at nearly the same time — sometimes under the same peak. Integration will count it as product.
Mass spectrometry does not care about hydrophobicity. It measures mass, and a missing residue changes mass by 57 to 186 daltons depending on which one. That difference is unmissable.
ESI: electrospray ionisation
The sample is dissolved and sprayed through a needle held at high voltage. The spray forms charged droplets; solvent evaporates until the peptide ions are released into the gas phase.
The characteristic feature is multiple charging. A peptide picks up several protons, so a 4000 Da molecule appears not at m/z 4000 but as a series of peaks — at roughly 2001 for [M+2H]2+, 1334 for [M+3H]3+, 1001 for [M+4H]4+ and so on. Software deconvolutes that series into one neutral mass.
Advantages: high accuracy, couples directly to HPLC so separation and identification happen in one run (LC-MS), and the multiple charging brings large molecules within reach of instruments with limited m/z range. Disadvantages: sensitive to salts and buffers, and the raw spectrum is not readable without deconvolution.
MALDI: matrix-assisted laser desorption/ionisation
The sample is co-crystallised with a small organic matrix compound that absorbs at the laser’s wavelength. A laser pulse hits the spot; the matrix absorbs the energy and vaporises, carrying the peptide with it and transferring a proton.
The characteristic feature is predominantly single charging. A 4000 Da peptide appears at m/z ≈ 4001. The spectrum is directly readable with no deconvolution.
Advantages: far more tolerant of salts and buffers, simple spectra, fast, works well on mixtures. Disadvantages: usually lower mass accuracy than modern ESI instruments, and it does not couple to liquid chromatography in the same seamless way.
Choosing between them
| ESI | MALDI | |
|---|---|---|
| Charge states | Multiple | Mostly single |
| Couples to HPLC | Yes, directly | Not directly |
| Salt tolerance | Low | High |
| Spectrum readability | Needs deconvolution | Direct |
| Typical use | Routine QC, LC-MS purity plus identity | Rapid confirmation, salty samples |
For research peptide certificates, ESI in an LC-MS configuration is the most common, because it produces purity and identity from a single injection.
Reading the result
The certificate should state a theoretical mass — calculated from the sequence — and an observed mass. They should agree closely; for a well-run instrument, within a fraction of a dalton is normal, and agreement to the nearest whole dalton is acceptable for routine QC.
Common informative discrepancies:
- +16 Da — oxidation, typically of methionine
- +1 Da — a free acid where a C-terminal amide was expected, or deamidation
- −18 Da — loss of water, or an unexpected cyclisation
- −57 to −186 Da — a deletion sequence, the mass of the missing residue
- Roughly double the expected mass — a dimer, often disulfide-linked
A certificate showing purity with no mass confirmation leaves the central question unanswered. On a long or modified peptide, that omission matters more than a percentage point of purity.
Our purity verification guide covers both methods together, and the certificate guide covers the document as a whole.
All products and information referenced are for in-vitro research and laboratory use only. Nothing here is medical advice, and no therapeutic claim is made or implied.