Mass Spectrometry for Peptide Identification

Electrospray ionisation produces multiply charged ions, so a peptide of mass 4000 appears at m/z values well below 4000 depending on charge state. The instrument measures mass-to-charge, and the neutral mass is deconvoluted from a series of charge states. A spectrum showing several peaks is usually one compound at several charge states, not several compounds.

MALDI tends to produce singly charged ions and tolerates salts and buffers better, which makes it convenient for quick identity checks on less clean samples. ESI couples directly to liquid chromatography, which is why LC-MS dominates where separation and identification are both needed.

Monoisotopic mass uses the lightest isotope of each element; average mass uses natural isotopic abundance. For small peptides the two differ by a little; for larger ones the difference is several daltons and matters when comparing against a calculated value.

A reported mass that appears a few daltons off is frequently a monoisotopic-versus-average mismatch rather than a real discrepancy. It is worth checking which the certificate used before concluding anything.

Selecting a precursor ion and fragmenting it produces b and y ion series whose spacings correspond to residue masses, allowing the sequence to be read directly. This distinguishes isomers and positional variants that intact mass cannot.

Two residue pairs remain genuinely hard: leucine and isoleucine are identical in mass, and lysine and glutamine differ by a very small amount requiring high resolution. Knowing these limits prevents overclaiming from a spectrum.

Deconvolution software turns a charge-state envelope into a neutral mass, and it will produce an answer whether or not the input justified one. Artefacts are common: a noisy envelope, overlapping species or an incorrect charge assignment can yield a confident-looking mass that is wrong.

The practical defence is to look at the raw charge-state series rather than only the deconvoluted output. A genuine species produces a coherent ladder of charge states with consistent spacing. A mass reported without the underlying spectrum is an assertion, which is one reason a certificate that includes only a number is weaker evidence than one that includes the trace.

Adducts are a related trap. Sodium and potassium adducts shift the observed mass upward in characteristic increments, and a peptide that looks twenty-two daltons heavy is frequently a sodium adduct rather than a modified molecule.

It is not inherently quantitative without a suitable internal standard, because ionisation efficiency varies between compounds and with matrix. This is exactly the problem isotopically labelled internal standards solve: a heavy-labelled version of the analyte behaves identically through preparation and chromatography while resolving by mass.

And it cannot see what does not ionise. Counter-ions, water and some non-peptide residues contribute to vial mass while being effectively invisible, which is why mass spectrometry needs to sit alongside content and water determination rather than replacing them.