Confirming identity by mass spectrometry

A peptide's sequence fixes its elemental formula, and the formula fixes a theoretical mass. Mass spectrometry measures the real mass and compares it with that prediction, within the mass accuracy of the instrument.

How a peptide mass is measured

Two ionization methods dominate. Electrospray ionization (ESI) sprays a solution of the peptide into a charged mist and produces multiply charged ions, written [M+nH]n+, each recorded as a mass-to-charge ratio (m/z). The series of charge states is deconvoluted: every peak yields the same molecular mass M, and agreement across the series is a consistency check.

Matrix-assisted laser desorption/ionization with a time-of-flight analyzer (MALDI-TOF) fires a laser at the peptide co-crystallized with a matrix. It gives mostly singly charged ions, so one dominant peak sits near M + 1.007 Da. Either way, the measured mass is compared with the theoretical mass calculated from the sequence.

Monoisotopic versus average mass

Hydrogen, carbon, nitrogen, oxygen and sulfur each occur as several stable isotopes, so one peptide appears as a cluster of peaks, the isotopic envelope. The monoisotopic mass uses only the lightest isotope of every element; the average mass weights each isotope by natural abundance.

Small peptides are reported as monoisotopic because the lightest peak is resolved and easy to identify. As size grows, the envelope widens and the lightest peak shrinks until it is too small to measure reliably, so large peptides and proteins are reported as average mass. For a peptide of roughly 4 to 5 kDa the two differ by about 2.5 to 3 Da (roughly 0.06% of the mass), so a stated mass needs its type named.

What a matching mass establishes

A match shows that the measured mass is consistent with the intended amino acid composition. Nothing more follows from it:

• Order. Sequence isomers, with the same residues in a different order, have identical masses.
• Chirality. D- and L-amino acids have identical masses. Leucine and isoleucine, too, have identical mass, so a swap between them is invisible to an intact mass measurement.
• Purity. Peak height reflects ionization efficiency as well as amount, so a spectrum is not a purity figure without calibrated standards.
• Counter-ions. Acetate or trifluoroacetate salts dissociate in the source, so the measured mass is that of the peptide alone.

How sequence is confirmed

When order matters, tandem mass spectrometry (MS/MS) selects one peptide ion, fragments it along the backbone and measures the pieces. Cleavage yields b ions, which retain the N-terminus, and y ions, which retain the C-terminus. Consecutive ions in a ladder differ by the mass of one residue, so the sequence is read from the spacing.

Edman degradation removes and identifies residues one at a time from the N-terminus. It suits short chains, needs a free N-terminus and, unlike an intact mass measurement, can separate leucine from isoleucine.

Where Solara stands

Solara does not publish mass spectra for any lot. Where a product page states an identity result, it is the manufacturer's laboratory comparing its lot with a reference standard by HPLC, not a mass spectrum, and it describes that lot, not an individual vial. A label names the compound; a mass spectrum is evidence for that name and says nothing about the quantity in milligrams.