Key facts
- x-axis
- Mass-to-charge ratio (m/z)
- y-axis
- Relative abundance, with the base peak set to 100%
- Base peak
- The most intense peak in the spectrum
- Isotope peak spacing
- About 1.003 divided by the charge (z) on the m/z axis
- Common peptide ions
- [M+H]+, [M+2H]2+, [M+3H]3+
- Common adducts
- [M+Na]+ (+21.98 from [M+H]+), [M+K]+ (+37.96)
What is a mass spectrum?
A mass spectrum is what a mass spectrometer produces: a graph with mass-to-charge ratio along the horizontal axis and ion abundance up the vertical axis [1].
Every peak marks ions that reached the detector at a particular m/z. You never see molecules directly on a mass spectrum. You see ions, and those ions might carry one charge or several, might have picked up extra atoms during ionization, or might be fragments of the original molecule. Reading the spectrum is detective work: tracing each ion back to the molecule it came from. For how the instrument generates the data, see how mass spectrometry works; for the technique as a whole, see what is mass spectrometry.
How do you read the axes of a mass spectrum?
Along the bottom runs m/z, which is the ion's mass in daltons divided by its number of charges. Up the side runs signal intensity, usually normalized so that the tallest peak reads 100%.
Since the x-axis is a ratio, a peak at m/z 1,001 might be a singly charged ion of about 1,000 Da or a doubly charged ion of about 2,000 Da. The y-axis is relative, too. A peak at 40% is 40% as intense as the base peak; it is not 40% of the sample. Some spectra show absolute counts instead. Those help you judge signal strength, but they still won't let you compare amounts of different molecules, because each one ionizes with its own efficiency.
| Term | Meaning |
|---|---|
| m/z | Mass of the ion divided by its charge number |
| Base peak | Tallest peak, set to 100% relative abundance |
| Precursor or molecular ion | Ion representing the intact molecule, such as [M+H]+ |
| Charge state (z) | Number of charges carried by the ion |
| Isotope envelope | Cluster of peaks from molecules containing heavier isotopes |
| Adduct | Ion formed with an extra atom or molecule, such as sodium |
| Fragment ion | Ion produced by breaking the precursor, as in MS/MS |
What is the base peak in a mass spectrum?
The base peak is simply the most intense peak in the spectrum. Every other peak is scaled against it.
Don't assume the base peak is your molecule. In electrospray spectra of peptides it is often one particular charge state, say [M+2H]2+, while the [M+H]+ ion is small or missing altogether. It could also be a solvent cluster, a background contaminant or a fragment. Use the base peak as the scaling reference first, and then figure out what it actually is.
What do isotope peaks show?
Isotope peaks come from molecules of the same compound that happen to contain one or more heavier isotopes, mostly carbon-13. How far apart they sit tells you the ion's charge.
About 1.1% of natural carbon is carbon-13. A peptide with dozens of carbon atoms will therefore always include some molecules that are one, two or three daltons heavier than the all-carbon-12 form, and on a high-resolution spectrum they show up as a cluster of peaks. The first peak in that cluster, made from the lightest common isotope of every element, is the monoisotopic peak. The bigger the molecule, the smaller a share of the cluster the monoisotopic peak becomes. For large biomolecules it can be hard to spot at all, so software often fits the whole isotope distribution to find it [2].
Spacing gives away the charge. Neighboring isotope peaks differ in mass by about 1.003 Da, which puts them 1.003/z apart on the m/z axis: about 1.0 for a singly charged ion, 0.5 for a doubly charged ion and 0.33 for a triply charged one.
The molecular weight on a product page is an average mass, weighted by natural isotope abundance. A high-resolution mass spectrum reports monoisotopic masses. For a peptide around 1,400 Da the two differ by roughly one dalton, and the gap widens as molecules get bigger. Compare like with like.
How do you work out the charge state and neutral mass?
First find the charge state, either from isotope spacing or from a series of related peaks. Then get the neutral mass by multiplying m/z by the charge and subtracting the mass of the protons that were added.
For a protonated ion, m/z = (M + z × 1.007) / z, where M is the neutral mass and 1.007 Da is the mass of one proton. Rearranging gives M = z × (m/z) − z × 1.007. Electrospray usually produces several charge states of the same molecule [3], so one peptide shows up more than once. If those peaks all give the same M, that's strong evidence they belong together.
| Ion | Charge (z) | Expected m/z | Isotope spacing |
|---|---|---|---|
| [M+H]+ | 1 | 2,001.01 | 1.00 |
| [M+2H]2+ | 2 | 1,001.01 | 0.50 |
| [M+3H]3+ | 3 | 667.67 | 0.33 |
| [M+4H]4+ | 4 | 501.01 | 0.25 |
Software does this calculation for you in a step called deconvolution, which collapses the series of charge states into one neutral mass. When a report gives both the observed m/z values and the deconvoluted mass, you can check the arithmetic yourself.
What are adducts and other unexpected peaks?
An adduct is an ion in which the molecule has picked up something besides a proton, usually sodium or potassium. Adducts turn up at predictable offsets from the main ion.
| Offset from [M+H]+ | Likely explanation |
|---|---|
| +21.98 | Sodium adduct, [M+Na]+ |
| +37.96 | Potassium adduct, [M+K]+ |
| +15.99 | One extra oxygen atom, often an oxidized methionine |
| −18.01 | Loss of water |
| Minus one residue mass | Deletion sequence, a common synthesis-related impurity |
With multiply charged ions, divide each offset by the charge. Treat offsets as clues rather than conclusions. A mass difference that fits oxidation or a missing residue gives you a hypothesis to test with chromatography or MS/MS, and the same shift sometimes has more than one explanation. Reviews of peptide impurity profiles list the deletion, truncation, oxidation and protecting-group impurities these offsets most often point to [4].
What is an MS/MS spectrum?
An MS/MS spectrum, also called a product-ion spectrum, shows the fragments you get when one selected ion is broken apart. You read it to confirm a sequence, not an intact mass.
Peptides fragment mainly into two series: b ions, which hold the N-terminus, and y ions, which hold the C-terminus [5]. Within a series, neighboring peaks are one amino acid's residue mass apart, so the spacing spells out the sequence, reading in one direction or the other [6]. There is one well-known gap. Leucine and isoleucine have identical residue masses (113.08 Da), and standard low-energy fragmentation can't tell them apart. Keep that in mind whenever you read a sequence claim.
What should a mass spectrum on a COA show?
If a mass spectrum is attached to a certificate of analysis, it should include the spectrum itself and state the observed mass, the theoretical mass and which batch it belongs to.
- The ionization method (ESI or MALDI) and the instrument type.
- The observed m/z values and the charge state assigned to each.
- The calculated neutral mass, the theoretical mass of the intended sequence, and the difference between the two.
- Whether the masses are monoisotopic or average.
- The batch or lot number and the date of testing.
A matching mass supports identity. It doesn't measure purity, because peak heights depend on how readily each species ionizes. Purity comes from HPLC data, as what HPLC purity means explains. The two fit together as described in identity vs. purity, and how to read a COA shows how they sit side by side on a real document.
FAQFrequently asked questions
What do the x-axis and y-axis of a mass spectrum show?
The x-axis is mass-to-charge ratio, written m/z: the ion's mass divided by its number of charges. The y-axis is intensity, usually given as relative abundance with the tallest peak set to 100%. Where a peak sits tells you which m/z was detected, and its height tells you how strong that signal was compared with the base peak.
Why does one peptide produce several peaks in a mass spectrum?
There are three main reasons. Electrospray attaches different numbers of protons, so the same peptide shows up at several charge states. Natural carbon-13 then gives each charge state its own cluster of isotope peaks. And some molecules pick up sodium or potassium in place of a proton, which produces adduct peaks at predictable offsets from the main ion.
What is the difference between m/z and molecular weight?
Molecular weight is the mass of the neutral molecule, normally an average based on natural isotope abundance. m/z is what the instrument actually measures, an ion's mass divided by its charge. To compare a spectrum with a molecular weight, convert the observed m/z into a neutral mass using the charge state, and check whether the reported value is monoisotopic or average.
Does a taller peak mean there is more of that compound?
Not necessarily. Peak height depends on how efficiently a molecule ionizes as well as on how much of it is there, and ionization efficiency varies a great deal. A trace of an impurity that ionizes easily can give a tall peak, while a larger amount of something that ionizes poorly can look minor. For quantitative comparisons you need calibration or a separate method such as HPLC.
What is deconvolution in mass spectrometry?
It's the calculation that turns a series of multiply charged ions into a single neutral mass. The software finds peaks that come from the same molecule at different charge states, works out each charge, and solves for the one mass that explains them all. What you get back is a simpler spectrum on a mass axis, much easier to compare with a theoretical molecular mass.
What is the monoisotopic peak?
It's the peak made by molecules built entirely from the most abundant isotope of each element: carbon-12, hydrogen-1, nitrogen-14, oxygen-16 and so on. It is the lightest peak in the isotope cluster. High-resolution reports usually quote monoisotopic mass, whereas product molecular weights are average masses, so don't expect the two figures to match exactly.
REFScientific references
-
Glish GL, Vachet RW. The basics of mass spectrometry in the twenty-first century. Nat Rev Drug Discov. 2003;2(2):140-50. PubMed 12563305
review -
Senko MW, Beu SC, McLafferty FW. Determination of monoisotopic masses and ion populations for large biomolecules from resolved isotopic distributions. J Am Soc Mass Spectrom. 1995;6(4):229-33. PubMed 24214167
analytical methods paper -
Fenn JB, Mann M, Meng CK, et al. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64-71. PubMed 2675315
original analytical method paper -
D'Hondt M, Bracke N, Taevernier L, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. PubMed 25044089
review -
Roepstorff P, Fohlman J. Proposal for a common nomenclature for sequence ions in mass spectra of peptides. Biomed Mass Spectrom. 1984;11(11):601. PubMed 6525415
nomenclature standard -
Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing. Nat Rev Mol Cell Biol. 2004;5(9):699-711. PubMed 15340378
review
Research Library
Testing & Analysis