Key takeaways
- Mass spectrometry measures molecular mass, which supports identity.
- Reports should list both expected and observed mass.
- ESI spectra show several charge states; MALDI usually shows singly charged ions.
- Identity and purity are separate questions that need separate tests.
A peptide can be highly pure and still be the wrong molecule, for example if a residue was skipped during synthesis. Mass spectrometry (MS) addresses that by measuring molecular mass.
Common ionization methods
- Electrospray ionization (ESI). Often coupled to HPLC (LC-MS). Peptides pick up several protons, so the spectrum shows a series of charge states that are deconvoluted to give the molecular mass.
- MALDI-TOF. The sample is mixed with a matrix and ionized by a laser. Peptides usually appear as singly charged ions, which makes spectra simple to read.
Expected versus observed mass
The expected mass is calculated from the sequence, including any modifications. Reports should state whether monoisotopic or average mass is used. Agreement between observed and expected mass, within the instrument’s accuracy, supports identity.
What MS can reveal
| Observation | Possible cause |
|---|---|
| Mass lower by one residue | Deletion sequence from an incomplete coupling |
| +16 Da | Oxidation, often of methionine |
| +22 Da or +38 Da | Sodium or potassium adducts |
| Incomplete removal of protecting groups | Mass higher by the protecting group |
Limits of MS identity testing
Mass alone cannot distinguish sequences with the same composition in a different order, or some isomers. For those questions, tandem MS (MS/MS) fragmentation or other methods are used.
Frequently asked questions
Does a mass match prove the sequence is correct?
It strongly supports it. Sequence-isomers need MS/MS or other methods to rule out.
Why do ESI spectra show several peaks?
Each peak is the same molecule carrying a different number of charges.
Is LC-MS a purity test?
LC-MS mainly supports identity. Purity is normally reported from the UV trace of HPLC.
Disclaimer. This article is for research and educational purposes only. It describes laboratory and analytical practice and is not medical advice. It contains no dosing, administration or human-use guidance. Veridryx products are sold for laboratory research use only and are not for human or veterinary use.
References
- ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.
- USP General Chapter <736> Mass Spectrometry. United States Pharmacopeia.