Mass Spectrometry Explained

ESI (electrospray) is the workhorse for soluble peptides — gentle, multi-charged ions. MALDI-TOF is preferred for higher-mass or aggregation-prone peptides and produces mostly singly charged ions.

ESI peptides typically appear as [M+H]+, [M+2H]2+, [M+3H]3+. Reconstructing the neutral mass from a charge envelope gives the most accurate molecular weight.

Identify the principal m/z, deconvolve to the neutral mass, and compare against the theoretical mass for your sequence. Tolerance: ±0.1 Da on a low-resolution instrument, ±0.005 Da on high-res.

A mass spectrometer measures mass-to-charge ratio; for singly-charged peptide ions that is effectively the molecular weight. The measured value is compared against the theoretical weight calculated from the sequence — and 'confirmed' means the two agree within the instrument's tolerance, not that they are identical to the last decimal.

Isotopic patterns are part of the check: a peptide's mass spectrum shows a cluster of peaks spaced by the mass difference between isotopes, and the spacing and shape of that cluster is a fingerprint that a single calculated number can't fake. Disulfide-linked or salt-adducted species shift the pattern in characteristic ways, which is why the raw spectrum — not just the summary value — is what a careful reviewer reads.

The two methods answer different questions, which is why a complete certificate uses both. HPLC separates what is present and quantifies how much of it is the main component; mass spectrometry identifies what that main component actually is. A pure-looking chromatogram can hide an impurity that co-elutes with the main peak; a mass spectrum alone cannot quantify purity at all.

The failure mode each method catches is the other's blind spot. Co-eluting impurities show up when mass spec reveals a second molecular species under one chromatographic peak; a mis-synthesised sequence shows up when the measured mass fails to match the theoretical one even though the HPLC trace looks clean. Paired, the two methods close most of the gaps either leaves open.