Numbers first: +2 · 4813.5 Da.
This should be a straightforward calculation and I keep getting two different answers.
The numbers are arbitrary; the method is what I am after.
Can someone walk through the arithmetic step by step?
Concretely, start from what electrospray ionisation does: it ionises the peptide without fragmenting it, creating singly or multiply charged species that the mass analyser then separates by their mass-to-charge ratio.
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
| Test | Answers | Does NOT answer |
|---|---|---|
| RP-HPLC, area % | What fraction of detected material is the target | How much target is present |
| Quantified content | Milligrams of peptide per vial | What the impurities are |
| ESI-MS identity | Whether the molecular weight matches | Purity, or isomeric substitution |
| Peptide mapping | Sequence, localised to a fragment | Quantity |
| Karl Fischer | Water content of the solid | Solvent content |
| LAL endotoxin | Pyrogen load in EU/mg | Sterility |
| Sterility test | Growth in defined media over 14 days | Endotoxin, or bioburden count |
In practice, 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.
Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.
Worth noting that source contamination is common and silent, so a result that looks too good to be true often is.
The practical summary: use mass spectrometry for identity, not for purity.
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Browse resultsOn the detail: tandem mass spectrometry fragments the ions and measures the fragment masses, which provides sequence information and is the best tool for confirming identity.
A mass shift of minus one hundred and twenty-eight usually means a missing Gln or Lys residue from a synthesis deletion sequence.
Specifically, 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.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
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.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
Concretely, scrambled disulfides have the same mass as correctly formed ones, so mass spectrometry alone cannot detect a scrambling failure.
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.
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.
False positives from contamination are common in mass spectrometry work, and running a blank between every sample and a solvent background are standard practice.
I would not trust a mass result without a good baseline and a blank injection check.
Always run a blank between samples and check for carry-over.
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