MALDI and ESI: Two Ways to Weigh a Peptide

A certificate reporting a measured mass does not usually say how the mass was obtained. It matters, because the two techniques in common use produce different spectra, resolve different things, and fail in different ways.

What each one does

MALDI — matrix-assisted laser desorption ionisation. The sample is co-crystallised with a small organic matrix compound, and a laser pulse vaporises and ionises it. The dominant product is the singly protonated molecule, so the spectrum is simple: one main signal, and the number next to it is close to the molecular mass plus one.

ESI — electrospray ionisation. The sample is sprayed from solution through a charged capillary, producing a series of ions carrying different numbers of protons. A single compound therefore appears as a ladder of peaks, each at a different mass-to-charge ratio, which software deconvolutes back to one mass.

Why the difference shows up on a report

A MALDI spectrum is easy to read and easy to publish. A single labelled peak, a number, done. That simplicity is also its limitation: because nearly everything arrives singly charged, the instrument must resolve small differences at the full mass of the molecule, and resolution generally degrades as mass rises.

An ESI spectrum is harder to read raw but carries more information. Charge states spread the molecule across a lower mass-to-charge range where resolution is better, which is why ESI on a decent instrument distinguishes a one-dalton difference on a peptide where MALDI may not. That one dalton is the difference between an amidated peptide and its free acid, and it is the deamidation shift. Deamidation and the 0.98 dalton shift covers why that number keeps appearing.

ESI also couples directly to liquid chromatography. An LC-MS run reports the mass of whatever is eluting at each moment, which is what makes it the decisive test for co-elution. MALDI is typically an offline spot measurement with no chromatographic dimension.

Where each one struggles

• MALDI can fragment labile modifications during desorption, and matrix signals crowd the low-mass region. Quantitation is unreliable, because signal depends heavily on how the sample crystallised.
• ESI is suppressed by salts and by involatile buffers. A sample carrying a heavy counter-ion load can give a poor spectrum for reasons that have nothing to do with the peptide. TFA and acetate counter-ions covers the salt form.

Neither technique is quantitative for purity in the way HPLC is. Signal intensity in mass spectrometry reflects how readily a species ionises, not how much of it is present, and two species in the same sample can ionise with very different efficiency. A mass spectrum confirms identity; the chromatogram carries the proportions.

Reading the report

Look for the measured mass beside a calculated mass for the sequence claimed, and check which form the calculation assumed. Look for enough of the spectrum to see whether satellites are present rather than a single cropped label. If the technique is named, it tells you how much confidence a one-dalton agreement deserves.

HPLC and mass spectrometry in purity verification covers how the two methods divide the work, and how to read a certificate of analysis covers the document as a whole.

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