Source of Mass spectrometry

{{Infobox method | name = Mass spectrometry | subtitle = Identification technique | image = mass-spectrum.svg | caption = 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 ionisation|Electrospray]] | Answers = What the molecule is; not how much unless calibrated }} '''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.{{r|aebersold2003}} A purity determination by [[High-performance liquid chromatography|chromatography]] establishes that one species dominates a sample; it does not establish which species. These are separate questions and require separate determinations. A [[Certificate of analysis|certificate]] reporting purity but no identity leaves the more fundamental of the two unanswered.{{r|usp1503}} Peptides ionised by [[Electrospray ionisation|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.{{r|aebersold2003,usp736}} == Ionisation and mass analysis == 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.{{r|aebersold2003}} 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|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.{{r|usp1503}} == What a mass confirms == | 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.{{r|aebersold2003}} Stereochemistry is invisible to mass spectrometry entirely. A D-amino acid substitution is isobaric with the L form and requires [[Amino acid analysis|chiral analysis]] to detect. For peptides synthesised with racemisation-prone residues this is a real rather than theoretical gap. == Quantification == Mass spectrometry is not inherently quantitative. Ionisation efficiency varies by orders of magnitude between molecules and is affected by the matrix, so peak intensity does not translate into concentration without calibration against a standard of the same substance.{{r|usp1503}} Where quantification is required, the usual approach couples chromatography to mass spectrometry — [[Liquid chromatography-mass spectrometry|LC-MS]] — and calibrates against a [[Reference standard|reference standard]], ideally with an isotopically labelled internal standard. This is standard practice in bioanalysis and is not typical of the certificates seen in research-chemical supply; a quantitative claim requires a validated procedure behind it.{{r|ich_q2}} The practical consequence is that a mass spectrum on a certificate answers "is this the right molecule" and nothing else. It does not report purity, does not report [[Peptide content|content]], and does not establish that minor species absent from the spectrum are absent from the material — poorly ionising impurities may simply not appear.{{r|aebersold2003}} == References == {{reflist}} <ref name="aebersold2003">Aebersold R, Mann M. "Mass spectrometry-based proteomics." ''Nature'' 422(6928):198–207 (2003). DOI:10.1038/nature01511. PMID 12634793.</ref> <ref name="usp1503">United States Pharmacopeia, General Chapter <1503>, ''Quality Attributes of Synthetic Peptide Drug Substances''.</ref> <ref name="usp736">United States Pharmacopeia, General Chapter <736>, ''Mass Spectrometry''.</ref> <ref name="ich_q2">International Council for Harmonisation, ''Q2(R2): Validation of Analytical Procedures'' (2023).</ref> == See also == * [[Electrospray ionisation]] * [[Liquid chromatography-mass spectrometry]] * [[Deamidation]] * [[Methionine oxidation]] * [[Amino acid analysis]] * [[Reference standard]] {{DEFAULTSORT:Mass spectrometry}} [[Category:Mass spectrometry]] [[Category:Analytical methods]] [[Category:Analytical science]]