Reading a mass spectrum

Mass is the most common way to establish identity (lesson 2). This lesson teaches you to read the spectrum itself: predict where a known peptide's peaks should sit and check whether a report's observed mass supports its identity claim. Why mass spectrometry catches swaps is covered in Identity: why it comes first.

What is on the axes of a mass spectrum?

The horizontal axis is m/z: the mass of an ion divided by the number of charges it carries. The vertical axis is relative intensity, usually scaled so the tallest peak is 100%. For peptides, m/z is not mass. A peptide carrying four extra protons appears at roughly a quarter of its mass.

Why would a peptide carry several charges? Electrospray ionisation (ESI), the usual source in LC-MS, "is unique among ionization techniques in that it produces multiply charged gas-phase macromolecular ions, whereas other ionization techniques typically produce singly charged ions" (Hogan et al., Analytical Chemistry, 2009). In a study of peptide charging, the average charge state under typical acidic conditions "was centered near the number of basic functional groups present within the peptide" (Meyer and Komives, J Am Soc Mass Spectrom, 2012). MALDI-TOF, by contrast, mostly gives singly charged ions, so its main peak sits close to the mass itself plus one proton. The method pages for LC-MS, MALDI-TOF and HRMS describe each instrument.

How do you calculate where a peptide's peaks should be?

Each charge in positive-mode ESI usually comes from an added proton. NIST gives the proton mass as 1.007276 u (NIST CODATA). For a peptide of mass M carrying z protons:

Apply it to tirzepatide, whose monoisotopic mass PubChem lists as 4810.5249 Da (PubChem). Retatrutide is 4728.4718 Da (PubChem, CID 171390338) and semaglutide 4111.1154 Da (PubChem).

If a vial labelled retatrutide gives a 4+ ion near 1203.6 rather than 1183.1, the 20.5 m/z gap at 4+ is the roughly 82 Da difference between tirzepatide and retatrutide divided by four. You do not need the software's deconvolution to see it.

How do you work out the charge of a peak?

Two ways. First, from the series: neighbouring charge states of the same molecule sit at m/z values that differ in a predictable way, and only one mass fits them all. Software does this automatically and reports a single deconvoluted mass. Second, from the isotope pattern, if the instrument resolves it: the peaks within one cluster are spaced roughly one mass unit apart divided by the charge, so a 4+ ion's isotope peaks are a quarter as far apart as a 1+ ion's.

Check a report's deconvoluted mass by reversing the formula on one of its listed peaks. Illustrative example: a report lists a 4+ ion at 1203.64. Then M = 4 x 1203.64 - 4 x 1.007276 = 4814.56 - 4.03 = 4810.53 Da, which matches tirzepatide's monoisotopic mass. The mass checker does this for any charge state you enter.

Monoisotopic or average mass: which one is the report using?

A peptide is not one exact mass. Most carbon is carbon-12, but a small fraction is the heavier carbon-13, and a molecule with over two hundred carbon atoms usually contains some. The monoisotopic mass assumes every atom is its lightest isotope. The average mass, the molecular weight, weights every isotope by its natural abundance. For tirzepatide, PubChem lists a monoisotopic mass of 4810.52 Da and a molecular weight of 4813 g/mol.

For tirzepatide the two bases differ by about 2.5 Da (4813 against 4810.52), so a gap of two or three Da on a peptide of this size can be just monoisotopic against average, not a wrong molecule. A gap of 82 Da cannot.

How close is close enough? Reading the error

Mass error is reported either in Da or in parts per million (ppm): ppm = (observed - expected) / expected x 1,000,000. Illustrative example: expected 4810.5249, observed 4810.5310. The difference is 0.0061 Da, which is about 1.3 ppm. What counts as acceptable depends on the instrument; high-resolution instruments are specified in low ppm, while lower-resolution ones are judged in Da. A report should say which tolerance it applied.

What else can appear in a spectrum?

• Adduct peaks. Ions carrying sodium or potassium instead of a proton, sitting a small, fixed step above the protonated peak. They come from salts in the sample or solvent and do not mean a different peptide.
• A second series. Another ladder of charge states that deconvolutes to a different mass. This can be an impurity, a degradation product or a second peptide. Its size matters: a minor series is a purity question; a dominant one is an identity question.
• Fragments. Pieces of the peptide broken in the source. In tandem MS they are made deliberately to read the sequence.

For mass spectrometry characterisation, the EMA guideline on synthetic peptides expects representative spectra and "tables with theoretical and observed mass values" (EMA, synthetic peptides guideline, 2025). That is a good model for any report: expected mass, observed mass, the difference, and the basis used.

Can peak height in a mass spectrum tell you how much is there?

Not reliably. Different molecules ionise with different efficiency, so a small impurity that ionises well can produce a tall peak while a larger component that ionises poorly produces a short one. Signal also shifts between charge states depending on the solvent and the instrument settings. A mass spectrum is the right tool for asking what is there. It is the wrong tool for asking how much.

That is why LC-MS and HPLC-UV work as a pair: the chromatogram gives the proportions, the spectrum gives each peak a name. If a spectrum shows a second series, check the size of the matching chromatogram peak before deciding whether it is a trace impurity or a real problem. Next, the microbiological questions: Endotoxin and sterility.

Check your understanding

1. A peak sits at m/z 1028.79 and you know it is 4+. What mass does it correspond to? 4 x 1028.79 - 4 x 1.007276, about 4111.13 Da, matching semaglutide's monoisotopic mass.
2. A report gives expected 4813.0 and observed 4810.5 for tirzepatide. Is identity in doubt? Probably not. The expected figure is the average mass and the observed is monoisotopic; the 2.5 Da gap is the basis difference.
3. Why does a peptide appear as several peaks in an ESI spectrum? ESI gives it several different numbers of charges, and each charge state appears at a different m/z.