Conditions: +5 · 4113.6 Da.
I want the working, not the result — I need to be able to redo it with different numbers.
I care about the precision as well as the value — I want to know how many figures are real.
Where is my error, and what is the correct working?
m/z = 823.73 at 5+. Electrospray charges a peptide by adding protons, so the observed ion is the neutral mass plus 5 protons, all divided by the charge: (4113.6 + 5 × 1.00728) ÷ 5 = 4118.636 ÷ 5 = 823.73. 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 686.61, 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.
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.
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 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.
A correct mass is necessary for identity but not sufficient — you also need the chromatography to confirm it.
Analytical standards and reagents with traceable certificates. Every quantitative result you read inherits the accuracy of the standard behind it.
Shop standardsSpecifically, 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.
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.
Electrospray ionisation soft-ionisation behaviour is well-characterised and standards exist for m/z calibration and mass accuracy assessment.
Always run a blank between samples and check for carry-over.
On the detail: 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 mass shift of minus one hundred and twenty-eight usually means a missing Gln or Lys residue from a synthesis deletion sequence.
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.
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.
Put another way, 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.
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 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.
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.
edited 16 Sept 2024 by lane_transit — added the method parameters
The limit of detection in electrospray is orders of magnitude better than in HPLC, which means a mass spectrometer can see species that the chromatograph cannot.
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 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.
I would not trust a mass result without a good baseline and a blank injection check.
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.