Mass Spectrometry for Peptide Identity: A Practical Guide

What the instrument measures

A mass spectrometer measures mass-to-charge ratio, written m/z. In electrospray ionization the peptide acquires protons in solution, so an ion with mass M and z protons has m/z = (M + z × 1.00728) / z. A small peptide such as a tripeptide mostly appears with one charge. A peptide of 3 to 4 kDa often carries three to six charges.

The instrument therefore reports several peaks for one molecule, each corresponding to a different charge state.

From m/z values to a molecular mass

To reach the intact mass, take two neighbouring peaks of the series, solve for their charges and compute the mass. Software does this routinely and is called deconvolution. A worked example: peaks at m/z 1001.0 and 501.0 are consistent with a mass of about 1000.0 Da, since 1001.0 × 1 − 1.007 ≈ 1000.0 and 501.0 × 2 − 2.015 ≈ 1000.0.

The deconvoluted mass is what is compared with the expected mass, and the report should show both the raw ions and the result.

Monoisotopic and average mass

Every element has natural isotopes, so a peptide is a family of molecules of slightly different mass. The monoisotopic mass uses the lightest isotope of each element. The average mass weights all isotopes by abundance. High-resolution instruments resolve the isotope peaks and report the monoisotopic mass, which is the first peak for small peptides.

For larger peptides the first peak is weak, and the instrument reports an average or most abundant mass. The two differ by about 1 Da at 1 kDa and about 2 Da at 4 kDa. The guide to average and monoisotopic mass explains how to calculate each.

Adducts, oxidation and other shifts

Not every peak is the intact peptide. Sodium and potassium from glassware and buffers produce adducts at +21.982 Da and +37.956 Da relative to the protonated ion. Methionine and tryptophan oxidize (+15.995 Da), asparagine and glutamine deamidate (+0.984 Da), and a reduced disulfide is 2.016 Da heavier than the oxidized form.

Recognising these steps lets a reader decide whether a minor peak is a degradation product, an impurity or an artefact. The guide to degradation products and mass shifts lists them with examples.

• +21.982 Da: sodium adduct (replaces H with Na)
• +37.956 Da: potassium adduct
• +15.995 Da: one oxidation
• +0.984 Da: deamidation
• −18.011 Da: loss of water

What a good report shows

A report that supports identity gives the expected formula and mass, the instrument type and ionization mode, the observed ions and their charge states, the deconvoluted mass and the difference from the expected value. It states whether the mass is monoisotopic or average.

It also links the spectrum to the main HPLC peak, ideally by showing that the mass belongs to the retention time of the main component, so that the identity is not that of a minor impurity.

What mass spectrometry does not show

Two peptides with the same composition but a different sequence have the same mass, so an intact mass cannot tell them apart. Fragmentation (MS/MS) or amino acid analysis is needed for that. Mass spectrometry also does not measure how much peptide is present, and the signal strength of an ion depends on how well the molecule ionizes, so peak heights in a spectrum are not a purity measure.