The specifics, since they change the answer: +4 · 4813.5 Da.
I would rather understand the derivation than memorise the outcome.
Two people I asked gave two answers that differ by a factor of ten, which is suggestive.
Can someone walk through the arithmetic step by step?
m/z = 1204.38 at 4+. Electrospray charges a peptide by adding protons, so the observed ion is the neutral mass plus 4 protons, all divided by the charge: (4813.5 + 4 × 1.00728) ÷ 4 = 4817.529 ÷ 4 = 1204.38. 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 963.71, 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.
It helps to be literal here: 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.
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.
In practice, 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.
False positives from contamination are common in mass spectrometry work, and running a blank between every sample and a solvent background are standard practice.
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 BiochemA D-amino-acid substitution has the same molecular weight as the L-form, so mass spectrometry cannot distinguish them without fragmenting the peptide.
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.
Worth being precise here: 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.
Peptide mapping — enzymatic digestion followed by tandem mass spectrometry — can confirm the primary sequence and is the method of choice when identity is ambiguous.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
edited 29 Apr 2025 by dead_volume — corrected a unit error in the worked example
Scrambled disulfides have the same mass as correctly formed ones, so mass spectrometry alone cannot detect a scrambling failure.
A mass shift of minus eighteen usually means dehydration or a succinimide intermediate, which is pH-dependent and can be reversible.
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.
Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.
I would not trust a mass result without a good baseline and a blank injection check.
The practical summary: use mass spectrometry for identity, not for purity.
Identity confirmation from mass spectrometry means matching the observed m/z to the calculated m/z for your peptide at its known charge states.
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 ICH Q3A and Q3B thresholds for reporting, identification and qualification of impurities are the framework the pharmaceutical industry works to, and they are worth reading even though nothing in the research-grade supply chain is obliged to meet them, because they tell you which numbers a competent analyst would consider worth reporting at all.
One qualification: high-resolution mass spectrometry gives high mass accuracy but low speed, and the reverse is true for low-resolution instruments.
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.
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.
High-resolution mass spectrometry can distinguish a Lys-containing peptide from an Arg-containing peptide of similar mass because of the isotope difference.
The relevant pharmacopoeial reference points here are the general chapters on peptide purity and on bacterial endotoxins; both specify method validation requirements that research-grade certificates almost never claim to meet, and the absence of that claim is itself information.
Worth noting that source contamination is common and silent, so a result that looks too good to be true often is.
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.