Mass spectrometry is the method most often used to confirm the identity of a research peptide. On a certificate of analysis it usually appears as MS, LC-MS, ESI-MS or MALDI-TOF, followed by an expected mass, an observed mass and a short conclusion.
The technique measures one property precisely: the mass-to-charge ratio of ions produced from the sample. Everything on the identity line of a certificate follows from that single measurement, and so do its limits.
What the instrument actually measures
A mass spectrometer does three things in sequence. It converts molecules from the sample into gas-phase ions, it separates those ions according to their mass-to-charge ratio, written as m/z, and it counts how many arrive at the detector at each ratio. The output is a spectrum: signal intensity plotted against m/z.
Note that the axis is a ratio, not a mass. Recovering the molecular mass requires knowing the charge on the ion, which is where the shape of a peptide spectrum becomes relevant.
Ionisation methods
Two ionisation methods are common for peptides. Electrospray ionisation, written as ESI, sprays the sample from solution and tends to produce ions carrying several charges. Matrix-assisted laser desorption ionisation, usually paired with a time-of-flight analyser as MALDI-TOF, uses a laser pulse on a crystallised sample and commonly produces singly charged ions.
Why one peptide produces several peaks
A peptide analysed by electrospray usually appears not as one peak but as a series. Each peak corresponds to the same molecule carrying a different number of protons, written as [M+H]+, [M+2H]2+, [M+3H]3+ and so on. Because the charge increases while the mass stays the same, each successive species appears at a lower m/z.
An analyst uses this pattern to calculate the molecular mass, a step called deconvolution. A series of related peaks is therefore expected in a peptide spectrum and is not in itself evidence of multiple compounds.
Monoisotopic and average mass
Certificates sometimes report two expected masses. The monoisotopic mass is calculated using the most abundant isotope of each element. The average mass uses the natural isotopic mixture, and is slightly higher. Which one applies depends on the resolution of the instrument, so a small difference between an expected and observed figure may simply reflect which convention was used.
What a mass match shows
When the observed mass agrees with the mass calculated from the molecular formula of the named compound, within the tolerance of the instrument, the result is reported as conforming or consistent with the expected structure.
The precise claim is narrow and worth stating plainly: a species was detected whose mass matches the mass the named compound should have. That is meaningful evidence about identity, and it is the whole of what the measurement provides.
How close is close enough
Observed and expected masses rarely agree to every decimal place, so a result is assessed against a tolerance. High-resolution instruments express this in parts per million, where the allowed difference scales with the mass being measured. Lower resolution instruments are usually assessed against an absolute window in daltons.
A certificate that states the tolerance alongside the two figures is giving you what you need to judge the match yourself. Where no tolerance is stated, the conclusion line records the judgement of the analyst rather than a figure you can check.
What a mass match does not show
- Mass alone does not distinguish molecules that share a formula. Sequence isomers, built from the same residues in a different order, have the same mass.
- A mass measurement does not describe proportion. Detecting the expected species says nothing about what share of the material in the vial it represents, which is what HPLC is used to estimate.
- Ionisation efficiency varies between compounds, so peak intensity in a spectrum is not a reliable measure of relative amount.
Identity and purity are separate measurements that fail independently. Purity and identity testing covers why a certificate reports both, and why neither substitutes for the other.
Where sequence information comes from
Tandem mass spectrometry, written as MS/MS, addresses part of the isomer limitation. Ions of a selected mass are fragmented and the fragments are measured, and the pattern of fragment masses carries information about the order of residues. It is a separate analysis and appears on a certificate only when it was performed.
Reading a mass spectrometry section
- Which ionisation method is named, and which analyser?
- Is the expected mass monoisotopic or average?
- What is the difference between expected and observed, and is a tolerance stated?
- Is a spectrum included, so the reported figure can be checked against it?
- Was the measurement made on the same batch as the rest of the certificate?
How to read a certificate of analysis covers the document as a whole, and published certificates for compounds in our catalogue are on the certificates of analysis page.
All compounds and research materials supplied by PepX Research are for laboratory and research use only. They are not for human consumption.