Mass Spectrometry for Peptide Identity

What a mass spectrometer measures

A mass spectrometer does not weigh a molecule directly. It turns molecules into ions, sorts the ions, and reports their mass-to-charge ratio, written m/z. If an ion carries one charge, its m/z equals its mass; if it carries three charges, its m/z is about one third of its mass. Reading a spectrum is mostly a matter of working out which charge each peak carries.

Getting peptides into the gas phase

Peptides are fragile and polar, so the ionization method has to be gentle. Two methods dominate. In electrospray ionization a solution is sprayed through a charged needle and ions are released from the droplets. The characteristic result for large molecules is a series of peaks from ions of the same molecule carrying different numbers of charges.^[1] In matrix-assisted laser desorption ionization, the sample is mixed with a light-absorbing matrix and ionized by a laser pulse; it mostly produces singly charged ions, and early work showed it could handle proteins above 10,000 daltons.^[2]

Electrospray is easily connected to a liquid chromatograph, so the same run that separates the sample can report a mass for each peak. That pairing is called LC-MS.

Charge states: a worked example

For a positive-ion measurement, a peptide of neutral mass M that has picked up n protons shows at m/z = (M + n × 1.00728) ÷ n, where 1.00728 is the mass of a proton. Take a made-up peptide with a neutral monoisotopic mass of 1000.000 Da. Its singly charged ion appears at (1000.000 + 1.00728) ÷ 1 = 1001.007. The doubly charged ion appears at (1000.000 + 2.01456) ÷ 2 = 501.007, and the triply charged ion at (1000.000 + 3.02184) ÷ 3 = 334.341.

Going the other way, if two neighbouring peaks in a series are 501.007 and 334.341, the difference in charge between them is one, which fixes both charges and lets the neutral mass be worked out. Software does this automatically and is called deconvolution; it is still useful to know the arithmetic.

Average mass and monoisotopic mass

Elements come as isotopes. Carbon is mostly carbon-12, with about one atom in ninety carbon-13. A peptide with many carbons therefore appears as a cluster of peaks, each one dalton apart, rather than a single peak. The first peak of the cluster, made only of the lightest isotopes, gives the monoisotopic mass. The average mass weights every isotope by how common it is and sits near the centre of the cluster.

Below roughly 1,500 to 2,000 daltons the monoisotopic peak is usually the tallest in the cluster, and instruments with enough resolution report it. For larger peptides the tallest peak moves to heavier isotopes, and the monoisotopic peak becomes small, so average mass is often used instead. Models of isotope patterns exist to help find the right peak.^[3] What matters when comparing a measurement with a calculation is to compare like with like: monoisotopic to monoisotopic, average to average.

Other peaks in a spectrum

• Adducts. Sodium and potassium ions stick to peptides, giving peaks 21.982 and 37.956 above the protonated ion. They are normal, and a peak that fits these offsets is usually an adduct rather than a new substance.
• Oxidation. An oxidized methionine adds 15.995 daltons (one oxygen).
• Deamidation. Conversion of an asparagine side chain to aspartic acid adds 0.984 daltons, which is easy to miss at low resolution.
• Acetylation and amidation. An acetylated N-terminus adds 42.011 daltons. An amidated C-terminus changes the mass by −0.984 daltons relative to the free acid.
• Missing residues. A peptide that lacks one amino acid is lighter by exactly that residue’s mass. Differences like these are how related impurities are recognized.

Tandem mass spectrometry: reading the sequence

To go beyond a single mass, an ion can be selected, broken apart, and the fragments weighed. This is tandem mass spectrometry (MS/MS). In the gas phase, peptide ions most often break at the amide bonds along the backbone, giving two families of fragments called b ions and y ions. The mass difference between neighbouring fragments in a family equals the mass of one amino acid residue, so the sequence can be read off step by step.^{[4, 5]}

Fragmentation is not perfectly even. Cleavage is often stronger at certain residues, such as the bond next to aspartic acid, which is why real spectra do not always show a complete ladder.^[4] Software scores the match between observed and predicted fragments.

What a matching mass does and does not show

• It shows consistency. A mass within the instrument’s accuracy of the calculated value is consistent with the expected sequence. Accuracy is often stated in parts per million: for a 1,000 Da peptide, 5 ppm is 0.005 Da.
• It does not separate isomers. Leucine and isoleucine have identical masses. So do a peptide and its version with one L-amino acid replaced by its D-form, or aspartic acid and its isoaspartic form. Mass alone cannot tell these apart; fragmentation patterns, chromatography or other methods are needed.
• It is not quantitative by itself. Peak heights in a mass spectrum depend on how well each substance ionizes, so they are not a measure of how much is present.
• It is only as good as the calculation. Mass differences of a few daltons may come from the wrong charge state, the wrong isotope peak, a different salt, or an expected modification that was not included in the calculation.

Practical steps

1. Write down the exact sequence and any modifications such as an acetylated N-terminus, an amidated C-terminus or disulfide bonds.
2. Calculate both average and monoisotopic masses. The peptide molecular weight estimator gives both for linear sequences, as an estimate.
3. Work out the expected m/z for the charge states the instrument is likely to show.
4. Compare, allowing for adducts and for the stated accuracy of the instrument.
5. Record the instrument, the ionization mode, and the calculation you used, so the result can be repeated.

Where this fits

Chromatography answers “how much of the detected signal is one peak?” and mass spectrometry answers “is that peak the right mass?” Used together on the same run, they give a far more complete picture than either alone. See reading an HPLC chromatogram for the chromatographic side.