Mass Spectrometry and Peptide Identity

Mass spectrometry confirms a peptide's identity by measuring the mass-to-charge ratio (m/z) of its ions and comparing the molecular mass derived from them with the mass calculated from the expected molecular formula. Synthetic peptides are usually ionised by electrospray ionisation (ESI), which produces a series of multiply protonated ions, or by matrix-assisted laser desorption/ionisation (MALDI), usually with a time-of-flight analyser, which produces mostly singly protonated ions. Agreement within a stated tolerance shows that the main component has the intended composition; it does not measure purity.

What a mass spectrometer measures: m/z

A mass spectrometer does not weigh neutral molecules. It turns them into gas-phase ions, separates the ions by the ratio of their mass to their charge number, m/z, and records how many arrive at each value. The result is a spectrum: signal intensity against m/z.

Peptides are normally observed as protonated ions. A molecule of neutral mass M that picks up z protons forms the ion [M+zH]ᶻ⁺, which appears at:

m/z = (M + z × 1.00728) ÷ z

Here 1.00728 Da is the mass of a proton. For BPC-157, molecular formula C62H98N16O22 and monoisotopic mass 1418.704 Da, the first three charge states fall at:

The same molecule can therefore produce several peaks, and none of them sits at the neutral mass itself. Every reading of a spectrum starts by working out which ion each peak is.

Electrospray ionisation (ESI)

In ESI, the peptide solution flows through a fine capillary held at a high voltage. The liquid leaves as a spray of charged droplets, the droplets shrink as the liquid evaporates, and intact peptide ions pass into the gas phase. The process is gentle enough to keep large molecules whole. Fenn and colleagues showed in 1989 that it produces intact ions from large biomolecules, and it has been a standard method for peptides and proteins since.

The signature of an ESI spectrum is a series of peaks from one molecule carrying different numbers of protons, each differing from its neighbour by one charge. Larger peptides carry more charges, so even a 5 kDa peptide appears within the m/z range of routine instruments.

Because ESI works from a liquid stream, it couples directly to HPLC. In LC-MS, each chromatographic peak is measured as it leaves the column, which gives the mass of the main peak and of each impurity peak in one run.

Working out the charge state

Two adjacent peaks in an ESI series are enough to find the charge and the mass. Call the peak at lower m/z m₁ and the peak at higher m/z m₂; the m₁ ion carries one more proton. Then the charge of the m₂ ion is:

z = (m₁ − 1.00728) ÷ (m₂ − m₁)

The neutral mass is then M = z × (m₂ − 1.00728).

Take two ions of tirzepatide, calculated from its average molecular weight of 4813.45 g/mol: [M+5H]⁵⁺ at m/z 963.70 and [M+4H]⁴⁺ at m/z 1204.37. The charge of the second ion is (963.70 − 1.007) ÷ (1204.37 − 963.70) = 962.69 ÷ 240.67 = 4.00, and the mass is 4 × (1204.37 − 1.007) = 4813.45 Da. Deconvolution software applies the same arithmetic across the whole series and reports one neutral mass.

MALDI-TOF

In MALDI, the sample is mixed with a large excess of a small, UV-absorbing organic acid, the matrix, and dried on a metal target plate so that the two co-crystallise. A short laser pulse vaporises the matrix, which carries the peptide into the gas phase and transfers a proton to it. α-Cyano-4-hydroxycinnamic acid (CHCA) is a common matrix for peptides. MALDI produces mostly singly charged ions, so a peptide usually shows one main peak at its mass plus one proton.

The ions are then accelerated through the same electric potential into a field-free flight tube. Ions with a lower m/z travel faster, so the time each ion takes to reach the detector gives its m/z: flight time is proportional to the square root of m/z. A reflectron, an ion mirror at the far end of the tube, compensates for small differences in speed between ions of the same m/z, which sharpens the peaks.

Expected mass and observed mass

The expected mass comes from the molecular formula, which is fixed by the sequence and its modifications. The observed mass is what the instrument measures, converted from m/z to a neutral mass. A certificate should state both, and say which mass convention each uses.

On a high-resolution instrument, agreement is expressed as a mass error in parts per million (ppm): the difference between observed and expected mass, divided by the expected mass, multiplied by one million. For example, a BPC-157 [M+H]⁺ ion expected at m/z 1419.7114 and observed at 1419.7128 has an error of about +1.0 ppm. The observed figure is illustrative. Low-resolution measurements are compared within a wider tolerance, stated in daltons.

When the two masses disagree, the size of the difference is often informative. The values below are calculated from monoisotopic atomic masses.

Average mass and monoisotopic mass

Most elements have more than one stable isotope. Natural carbon contains 1.07% carbon-13, so a molecule with many carbon atoms exists as a mixture of isotopic forms. Its signal is a cluster of peaks about 1 Da apart, or about 1/z apart on the m/z scale for an ion of charge z, which also gives the charge state directly at high resolution.

• Monoisotopic mass uses only the most abundant isotope of each element: ¹²C, ¹H, ¹⁴N, ¹⁶O and ³²S. It matches the first peak of the isotope cluster.
• Average mass uses standard atomic weights, which average over natural isotopic abundance. It is the molecular weight listed in a specification and used for weighing, and it lies near the centre of the cluster.

The gap between the two grows with size, and so does the spread of the cluster. Relative peak heights here are calculated from NIST isotopic abundances.

For a small peptide, a high-resolution spectrum shows the monoisotopic peak clearly, and it is the natural point of comparison. For a larger peptide the monoisotopic peak is weak, so deconvoluted spectra are usually compared with the average mass. Mixing the conventions creates an apparent error of almost 1 Da for BPC-157 and about 3 Da for tirzepatide.

What mass spectrometry confirms, and what it does not

A matching mass is strong evidence that the main component has the intended elemental composition, including its modifications. A C-terminal amide, an acetylated N-terminus, a lipid side chain or a bound metal ion each change the mass by a known amount. Several things remain outside what a single mass measurement can show:

• Sequence order. Peptides with the same residues in a different order have the same mass. Tandem mass spectrometry (MS/MS) breaks the backbone into fragment ions, conventionally named b and y ions, from which the sequence can be read.
• Leucine and isoleucine. Their residue masses are identical, 113.08 Da, so mass alone cannot tell them apart.
• Near-isobaric residues. Glutamine (128.06 Da) and lysine (128.09 Da) differ by 0.036 Da, which only high resolution separates.
• Stereochemistry. D- and L-amino acids have the same mass, so a racemised impurity is invisible to a mass measurement.
• Purity. Different molecules ionise with different efficiency, so relative peak heights in a mass spectrum do not give a percentage.
• Counter-ions and water. The spectrum reports the peptide ion, not the composition of the solid.

Mass spectrometry and HPLC therefore answer different questions. Mass spectrometry asks whether the main component is the right molecule; HPLC asks how much of the separated material it accounts for. A certificate of analysis needs both. HPLC and peptide purity covers the second question, and how to read a peptide certificate of analysis shows where each result sits on a certificate. Peptide sequence notation explains how the sequence behind an expected mass is written, and m/z, average mass and monoisotopic mass are defined in the glossary.