How do I compute the +3 charge state m/z for a peptide of 3751.0 Da?

Setup, so nobody has to ask: +3 · 3751.0 Da.

The units are where I keep going wrong, so please be explicit about them.

I have sanity-checked the order of magnitude and it seems right, which is not the same as being right.

How many significant figures are actually justified here?

It helps to be literal here: two ions with the same nominal mass but different molecular formulae have different exact masses, and only high-resolution mass spectrometry can distinguish them.

A monoisotopic mass includes only the lightest isotope of each element, while the average mass weights by natural isotope abundance, and small peptides use monoisotopic mass.

More usefully, a mass shift of plus one usually means deamidation at asparagine or glutamine, which creates a secondary amine instead of an amide and changes the mass by exactly one.

Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.

The practical summary: use mass spectrometry for identity, not for purity.

Source contamination and carry-over between samples are the failure modes most specific to mass spectrometry, and they are invisible without a blank injection between samples.

Electrospray ionisation creates multiple charge states of the same peptide — a 4 kDa peptide might appear at +2, +3 and +4 — and all of them must be accounted for in the spectrum.

The m/z accuracy achievable depends on the mass analyser type — quadrupole gives low accuracy, time-of-flight gives moderate accuracy, and Orbitrap gives high accuracy.

False positives from contamination are common in mass spectrometry work, and running a blank between every sample and a solvent background are standard practice.

A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.

A mass shift of exactly zero with a shifted retention time points to an isomer — a scrambled disulfide or a racemised residue — which mass spectrometry alone cannot identify.

The charge state distribution depends on the solution pH, the structure of the peptide and the source conditions, so the same peptide can look different under different conditions.

The caveat is that a correct mass does not mean the peak is correct — isomers and co-eluting species can have the same m/z.

Always run a blank between samples and check for carry-over.

edited 20 Nov 2024 by Dr_Ingrid_Baumgartner — corrected a unit error in the worked example

Coupling HPLC to a mass spectrometer adds identity information to the chromatographic separation, but the mass spectrometer's ionisation conditions can distort the HPLC peak shape.

For a large peptide with multiple peaks in the mass spectrum, comparing the observed isotope pattern to the calculated pattern is a quick check that the formula matches.

The limitation is that mass spectrometry tells you the mass and almost nothing else, so it needs to be paired with chromatography or other identity information.

The practical summary: use mass spectrometry for identity, not for purity.