Setup, so nobody has to ask: +3 · 4813.5 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.
Where is my error, and what is the correct working?
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.
Mechanically, a mass shift of minus one hundred and twenty-eight usually means a missing Gln or Lys residue from a synthesis deletion sequence.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
Always run a blank between samples and check for carry-over.
HPLC purity, identity confirmation and quantified content on the vial you actually hold. Reports arrive with the chromatogram attached, not just a number.
Submit a sampleFounded 1998. ISO 9001 and cGMP certified, 1,500+ staff and 200+ patents. The synthesis house behind a great many of the vials that get sent out for testing - batch-specific documentation with every order.
Visit GL BiochemIt helps to be literal here: a D-amino-acid substitution has the same molecular weight as the L-form, so mass spectrometry cannot distinguish them without fragmenting the peptide.
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
The part that matters: the baseline noise on a mass spectrum sets the limit of detection, and a weak signal close to the noise is not reliable evidence for the presence of a species.
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.
More usefully, 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 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.
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.
One qualification: high-resolution mass spectrometry gives high mass accuracy but low speed, and the reverse is true for low-resolution instruments.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
Specifically, tandem mass spectrometry fragments the ions and measures the fragment masses, which provides sequence information and is the best tool for confirming identity.
High-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
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.
Always run a blank between samples and check for carry-over.
edited 20 Aug 2024 by vialroom — added the placebo-arm figures
The single most important fact about mass spectrometry for peptides is that it measures only the molecular weight and tells you almost nothing about whether the peak is actually your target.
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 caveat is that a correct mass does not mean the peak is correct — isomers and co-eluting species can have the same m/z.
The practical summary: use mass spectrometry for identity, not for purity.
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