How mass spectrometry confirms a peptide's identity

The identity line on a peptide COA usually reads something like "MS: conforms" with an observed and an expected mass. Behind that line is a measurement that weighs molecules, and a judgment about whether the weight fits the sequence.

What the instrument does

Every mass spectrometer does three things:

1. Ionizes the sample, giving molecules an electric charge.
2. Separates the ions by mass-to-charge ratio, written m/z.
3. Detects how many ions arrive at each m/z.

For peptides, the two common ways to ionize are:

Ionization	How it works	What the spectrum tends to look like
Electrospray (ESI)	Sample sprayed from a charged needle; droplets shrink until ions are released	Several peaks for one peptide, each a different charge state
MALDI	Sample mixed with a matrix and hit with a laser	Mostly a single peak at one charge

ESI is the one usually coupled to liquid chromatography, so it is the one most often behind an LC-MS line on a COA.

Why one peptide gives several peaks

In ESI a peptide picks up a varying number of protons. A peptide of mass M carrying 2 protons shows up at about (M + 2) / 2. With 3 protons, at about (M + 3) / 3. With 4, at about (M + 4) / 4. So one molecule produces a ladder of peaks, a charge-state envelope.

Software works backward from that ladder to a single neutral mass, a step called deconvolution. The deconvoluted mass is the figure that gets compared to the sequence.

A worked illustration with a made-up peptide of neutral mass 2,400.0 Da:

Charge	m/z observed (approx.)
2+	1,201.0
3+	801.0
4+	601.0

Each line points back to 2,400 Da.

Expected mass: monoisotopic or average

The expected mass comes from the sequence. There are two ways to state it:

• Monoisotopic mass uses the most abundant isotope of each element. High-resolution instruments resolve individual isotope peaks and report against this.
• Average mass weights every isotope by natural abundance. Lower-resolution measurements, and most catalog listings, use this.

The two differ by roughly a dalton or more for a peptide of a few thousand daltons. A report should say which it uses; comparing a monoisotopic observation to an average expectation can make a correct match look wrong.

What "conforms" means

The lab sets a tolerance, which depends on the instrument. A match inside that window is reported as conforming. The tolerance should be stated or at least implied by the instrument type.

What a match shows: the main species has the mass expected from the sequence.

What a match does not show:

• Purity. Ionization efficiency varies, so peak heights in a mass spectrum are not a reliable measure of amount.
• Sequence order. Two peptides with the same amino acids in a different order have the same mass. Distinguishing them takes fragmentation (MS/MS) or other methods.
• Counterions and water, which are not part of the peptide mass.

Reading a mass spectrum on a COA

• x-axis: m/z
• y-axis: relative intensity, with the tallest peak set to 100%
• labeled peaks, ideally with their charge states
• a stated observed (deconvoluted) mass, an expected mass, and whether it is monoisotopic or average

Common extra peaks

Small peaks a few units away from the main one are often adducts: the peptide carrying a sodium (+22 Da on the neutral mass) or potassium (+38 Da) ion instead of a proton. A peak 16 Da heavier can be an oxidized form. Peaks that do not fit any of these are worth asking about.