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Guide 03 · Identity

Mass spectrometry and identity

How a mass spectrum confirms a peptide is what its label says, and the limits of that confirmation.

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Why mass confirms identity

Every sequence has a calculable mass. If the measured mass matches the calculated one within the stated tolerance, the molecule in the vial very likely has the intended composition. A missing residue, an extra one or most unexpected modifications shift the mass well beyond a typical tolerance, but a smaller change can pass: deamidation, or a free acid where the sequence has an amide, adds only 0.98 Da, inside a ±1 Da tolerance.

Average versus monoisotopic

Monoisotopic mass uses only the most common isotope of each element; average mass weights every isotope by its natural abundance. High-resolution instruments resolve the isotope pattern and report monoisotopic values; lower-resolution data and most reference tables use average mass. Our product pages list both.

PeptideAverageMonoisotopicGap
BPC-15715 residues1419.5361418.70420.8318
MOTS-c16 residues2174.5922173.10771.4843
LL-3737 residues4493.2634490.57542.6876
Table 1. Both masses, in daltons, for three listed peptides, computed from their sequences. The gap grows with size, so a certificate should say which one it reports.

Charge states

Electrospray ionization (ESI) adds protons, so a peptide of mass M appears at one m/z for each charge state z. Larger peptides show several charge states; the instrument software deconvolutes them back to M. MALDI usually shows singly charged ions, [M+H]+.

Formula

M is the neutral monoisotopic mass, z the number of protons added (the charge) and 1.007276467 Da the mass of a proton.

Fig. 1 · ESI-MS, positive mode

Example values

Ionm/zMonoisotopic found, Da
[M+2H]2+710.361881001418.7092
[M+H]+1419.71648931418.7092

Every charge state gives back the same found mass: M = z × (m/z − 1.007276467).

Fig. 1. Example electrospray spectra, drawn from each sequence; they aren’t lot records.Example charge states for BPC-157, from its sequence’s mass with a small example error; not a lot record. Electrospray adds protons at basic sites (the N-terminus, lysine, arginine and histidine), so a larger peptide with more of them shows higher charge states.

What MS can’t see

Mass alone can’t distinguish leucine from isoleucine (identical mass), a D-amino acid from its L form (identical mass) or the order of residues in a sequence of the same composition. Those questions need HPLC comparison with standards, tandem MS or other methods, which is one reason purity and identity are reported together.