What I have: +2 · 4207.2 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 show the working rather than just the answer?
m/z = 2104.61 at 2+. Electrospray charges a peptide by adding protons, so the observed ion is the neutral mass plus 2 protons, all divided by the charge: (4207.2 + 2 × 1.00728) ÷ 2 = 4209.215 ÷ 2 = 2104.61. The proton term is the one people drop, and because it is z protons over z charges it shifts m/z by 1.007 at every charge state — small, and far larger than the mass accuracy of the instrument. The neighbouring charge state sits at 1403.41, and seeing the two of them where they belong is better identity evidence than either one alone. Use the average mass against an average-mass calculation and the monoisotopic mass against a monoisotopic one; mixing them costs you a couple of daltons on a peptide this size.
Put another way, 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.
A mass shift of minus one hundred and twenty-eight usually means a missing Gln or Lys residue from a synthesis deletion sequence.
| Δ mass (Da) | Most likely cause | Distinguishing feature |
|---|---|---|
| +1 | Deamidation (Asn or Gln) | New peak, slightly earlier retention |
| −17 | Loss of ammonia | Often with deamidation |
| −18 | Dehydration / succinimide | pH-dependent, reversible |
| +16 | Oxidation (Met, Trp) | Earlier retention, light-related |
| −128 | Missing Gln or Lys | Deletion sequence from synthesis |
| 0 | Isomer: racemisation or scrambling | Same mass, shifted retention |
To be exact about it, 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.
False positives from contamination are common in mass spectrometry work, and running a blank between every sample and a solvent background are standard practice.
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.
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 BiochemOn the detail: 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.
Deconvolution of a mass spectrum with multiple charge states produces a reconstructed neutral mass, and errors in the deconvolution produce errors in the inferred mass.
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.
Always run a blank between samples and check for carry-over.
Scrambled disulfides have the same mass as correctly formed ones, so mass spectrometry alone cannot detect a scrambling failure.
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.
More usefully, 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.
Worth noting that source contamination is common and silent, so a result that looks too good to be true often is.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
edited 20 Oct 2024 by mz_4113 — added the method parameters
Identity confirmation from mass spectrometry means matching the observed m/z to the calculated m/z for your peptide at its known charge states.
High-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
If you only pay for one test, pay for quantified content. Purity is the number everyone quotes and content is the number that changes what you do.
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 plus sixteen usually means oxidation at methionine or tryptophan, which is common in peptides and often comes from sample handling rather than synthesis failure.
In practice: ask for the chromatogram, check the method section, check the lot number against the vial, and set your accept threshold before you see the result rather than after.
Ask PeptideStack is a static archive. Posting is closed, but the norms are worth stating: answer the question that was asked, show your working, cite the trial or the certificate, and say plainly where the evidence runs out.