Mass spectrometry (revision 13)
Old revision·10:52, 11 Nov 2024·Chromatokid
| Mass spectrometryIdentification technique | |
|---|---|
Peptides ionise to multiply charged species; the charge envelope is deconvolved to a single mass. | |
| Measures | Mass-to-charge ratio of ions |
| Usual ionisation for peptides | Electrospray |
| Answers | What the molecule is; not how much unless calibrated |
| Analytical method infobox · conventions | |
Mass spectrometry measures the mass-to-charge ratio of ionised molecules. For peptides it is the ordinary means of confirming identity: an observed molecular mass agreeing with the calculated mass excludes most alternatives, and fragmentation can confirm the sequence itself.[1]
A purity determination by chromatography establishes that one species dominates a sample; it does not establish which species. These are separate questions and require separate determinations. A certificate reporting purity but no identity leaves the more fundamental of the two unanswered.[2]
Peptides ionised by electrospray carry multiple charges, so a single peptide produces a series of peaks at different charge states. The instrument software deconvolves this envelope into one neutral mass, and it is that deconvolved mass that appears on a report.[1][3]
Ionisation and mass analysis
[edit]Electrospray disperses a solution into charged droplets in an electric field; solvent evaporates and ions are released into the gas phase. It is a soft technique — the molecule survives largely intact — which is why it suits peptides and proteins. Matrix-assisted laser desorption is the other common soft method and tends to produce singly charged ions.[1]
Mass analysers differ in resolution and in mass accuracy. A quadrupole gives unit resolution and is adequate for confirming a mass to within a dalton; time-of-flight and orbital-trap analysers give sufficient resolution to determine a mass to a few parts per million, which distinguishes species a quadrupole cannot.
The distinction matters for peptide work because the confounders are close in mass. Deamidation adds 0.984 Da, which unit resolution cannot resolve from the parent; oxidation adds 16 Da, which it can. A report stating an observed mass without stating the resolution of the instrument understates the ambiguity.[2]
What a mass confirms
[edit]| Observation | Excludes | Does not exclude |
|---|---|---|
| Mass agrees with calculated | Most substitutions and truncations | Isobaric variants; stereochemistry |
| Mass differs by +16 Da | — | Methionine oxidation |
| Mass differs by +0.98 Da | — | Deamidation |
| Fragmentation matches sequence | Isobaric rearrangements at resolved positions | Nothing at unresolved positions |
Intact-mass agreement is a strong but incomplete identification. Two different sequences composed of the same residues have identical masses, and inverting the order of two residues changes nothing about the intact mass. Only fragmentation — tandem mass spectrometry, which selects a precursor ion, breaks it and measures the fragments — addresses sequence order.[1]
References
- ^ a b c d Aebersold R, Mann M. "Mass spectrometry-based proteomics." Nature 422(6928):198–207 (2003). DOI:10.1038/nature01511. PMID 12634793.
- ^ a b United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.
- ^ United States Pharmacopeia, General Chapter <736>, Mass Spectrometry.