Key facts
- Core measurement
- Mass-to-charge ratio (m/z) of ions
- Four stages
- Sample introduction, ionization, mass analysis, detection
- Peptide ionization methods
- Electrospray ionization (ESI) and MALDI
- Common mass analyzers
- Quadrupole, time-of-flight, ion trap, Orbitrap
- Operating environment
- High vacuum inside the analyzer
- Output
- Mass spectrum: ion abundance plotted against m/z
How does mass spectrometry work? The short answer
A mass spectrometer weighs molecules indirectly. First it gives them an electric charge. Then electric or magnetic fields sort the resulting ions by mass-to-charge ratio, and the instrument records how many ions land at each value [1][2].
A benchtop quadrupole and a high-resolution Orbitrap look nothing alike, yet both run through the same four stages. The designs differ in how each stage is done and in how finely they can tell two masses apart. Definitions and core terms live on the companion page, what is mass spectrometry. Here we take a peptide sample through the instrument stage by stage.
| Stage | What happens | Peptide example |
|---|---|---|
| 1. Sample introduction | The sample enters as a liquid stream or a dried spot | Peptide solution infused directly or eluting from an HPLC column |
| 2. Ionization | Neutral molecules gain or lose charge | ESI adds protons, giving [M+2H]2+, [M+3H]3+ and so on |
| 3. Mass analysis | Ions are separated or filtered by m/z | A quadrupole, time-of-flight tube or Orbitrap resolves the ions |
| 4. Detection and data processing | Arriving ions are counted and converted into a spectrum | Software plots abundance against m/z and calculates the neutral mass |
Step 1: How does the sample get into the instrument?
Depending on the ion source, the sample enters the mass spectrometer either as a continuous liquid stream or as a dried spot on a target plate.
Electrospray needs the peptide in a volatile solvent, typically water and acetonitrile with a little formic acid. It is pumped in directly or fed from a liquid chromatography column. That second arrangement, LC-MS, spreads components out in time before they are ionized, which keeps the spectrum simpler at any one moment and cuts down on molecules competing for charge. MALDI is different: the peptide is mixed with a light-absorbing matrix, dried onto a metal plate and loaded into the source.
What else is in the sample counts here. Non-volatile salts, detergents and high levels of certain ion-pairing agents can drown out the signal of the molecule you care about. This is called ion suppression, and it's one reason LC-MS methods lean on volatile buffers and additives.
Step 2: How are molecules turned into ions?
Only charged particles can be steered and sorted by electric and magnetic fields, so the ion source, the front end of every mass spectrometer, has one job: turning molecules into ions.
Electrospray ionization (ESI)
In ESI, the liquid sample sprays from a fine needle held at high voltage and breaks into a mist of charged droplets. The solvent evaporates, the droplets shrink, and eventually charged analyte ions escape into the gas phase. In 1989, Fenn and colleagues showed that this gentle process could carry large, intact biomolecules into a mass spectrometer, often with several charges on each molecule [1]. So a peptide of neutral mass M shows up as a series of ions: [M+H]+, [M+2H]2+, [M+3H]3+ and so on.
Matrix-assisted laser desorption/ionization (MALDI)
MALDI fires a pulsed laser at the dried mix of matrix and sample. The matrix soaks up the energy and helps pass charge to the analyte, giving mostly singly charged ions. Karas and Hillenkamp reported in 1988 that the approach could ionize proteins with masses above 10,000 daltons [3]. Because most peaks are [M+H]+, MALDI spectra are often easier to read.
Both count as soft ionization techniques, since most molecules come through intact. Without that gentleness, a mass spectrometer could only weigh a peptide's fragments, never the whole molecule.
Step 3: How does the mass analyzer separate ions?
Inside the mass analyzer, the core of the mass spectrometer, electric fields, magnetic fields or a combination separate ions by mass-to-charge ratio. This happens under high vacuum so the ions don't bump into gas molecules along the way [2].
| Analyzer | Principle | Typical use with peptides |
|---|---|---|
| Quadrupole | Oscillating voltages on four rods let only a chosen m/z pass at a time | Targeted measurement and selecting ions before fragmentation |
| Time-of-flight (TOF) | Ions get the same energy push; lighter ions reach the detector sooner | Fast acquisition and a wide mass range; common with MALDI |
| Ion trap | Ions are held in an electric field and ejected by m/z | Repeated isolation and fragmentation |
| Orbitrap | Ions orbit a central electrode; their oscillation frequency reveals m/z | High resolution and high mass accuracy |
Resolution and accuracy: rating a mass spectrometer
When you read results, two performance figures count. Resolution is how well the analyzer pulls apart two nearby m/z values. Push it high enough and a peptide's individual carbon-13 isotope peaks come into view. Mass accuracy is how close the measured value lands to the true one, usually given in parts per million (ppm). High-resolution analyzers such as the Orbitrap can get within a few ppm, which drastically shortens the list of molecules that could explain a peak [4].
Step 4: How is the signal detected and turned into a spectrum?
At the detector, each ion arrival becomes an electrical signal. The data system turns that signal into a plot of abundance against m/z, which is the mass spectrum.
Most quadrupole and time-of-flight instruments use an electron multiplier, which amplifies every ion impact into a current you can measure [2]. The Orbitrap mass spectrometer works another way. It picks up the image current induced by ions as they oscillate, then converts those frequencies into m/z mathematically [4]. Software then labels peaks, groups isotope peaks and assigns charge states. For multiply charged electrospray data it also works out the neutral mass, a step called deconvolution. Reading the finished plot is covered in what is a mass spectrum.
How does tandem mass spectrometry (MS/MS) work?
Tandem mass spectrometry picks out one ion, breaks it into fragments and measures those fragments in a second round of mass analysis, usually inside the same mass spectrometer.
With peptides, colliding the selected ion with an inert gas tends to snap the backbone at the peptide bonds. Fragments that hold on to the N-terminus are b ions; those that keep the C-terminus are y ions, following a nomenclature Roepstorff and Fohlman proposed in 1984 [5]. Neighboring ions in a series differ by the mass of a single amino acid residue. Read the ladder of fragment masses and you can rebuild part or all of the peptide sequence [6], which is much stronger identity evidence than an intact mass by itself.
What does mass spectrometry tell you about a peptide sample?
What a mass spectrometer gives you is the masses of the ions present. That can confirm the sample contains a molecule of the expected mass, and it can expose species whose masses you didn't expect.
- Identity evidence: an observed mass that matches the theoretical mass of the intended sequence within the instrument's accuracy.
- Impurity clues: peaks at masses consistent with a missing residue, an oxidized methionine (+16 Da) or a protecting group that was not removed.
- Sequence evidence: MS/MS fragment ladders consistent with the expected order of residues.
On its own, though, mass spectrometry doesn't measure purity. Molecules ionize with different efficiencies, so a tall peak doesn't necessarily mean a lot of that component. That's why mass spectrometry and HPLC usually appear side by side on a report. HPLC vs. mass spectrometry lays out the comparison, and peptide testing shows how both feed into a peptide's documentation.
FAQFrequently asked questions
What are the main parts of a mass spectrometer?
Four functional parts: a sample inlet, an ion source that turns molecules into ions, a mass analyzer that separates those ions by mass-to-charge ratio, and a detector that counts them as they arrive. A vacuum system keeps gas out of the analyzer, and a data system turns detector signals into a mass spectrum. Instruments mostly differ in their ion source and analyzer.
Why does mass spectrometry need a vacuum?
Inside a mass spectrometer, ions have to get from the source, through the analyzer, to the detector without hitting gas molecules. A collision would scatter an ion, alter its path and energy, and smear the separation by mass-to-charge ratio, so analyzers run under high vacuum. Electrospray sources sit at atmospheric pressure, which is why instruments use several pumping stages to move ions from the open source into the evacuated analyzer.
What is the difference between ESI and MALDI?
Electrospray sprays a liquid sample from a charged needle and usually gives multiply charged ions, which makes it a natural partner for liquid chromatography. MALDI uses a laser to desorb a sample mixed with a matrix on a plate and gives mostly singly charged ions. Both are soft and keep peptides intact; they just fit different workflows and instruments.
Does mass spectrometry measure mass or mass-to-charge ratio?
Mass-to-charge ratio, written m/z, rather than mass itself. Picture a peptide with a neutral mass of 2,000 Da carrying two extra protons: it shows up near m/z 1,001. Software figures out the charge state, either from the spacing of isotope peaks or from a series of related peaks, and uses it to calculate the original molecule's neutral mass.
Can mass spectrometry tell how pure a peptide is?
Not reliably, at least not alone. Molecules ionize with different efficiencies, so peak height in a mass spectrum doesn't tell you how much of a component is there. Where mass spectrometry shines is identity and catching unexpected species. Peptide purity percentages normally come from HPLC with UV detection, often paired with mass spectrometry as LC-MS.
How accurate is mass spectrometry for peptides?
That depends on the analyzer inside the mass spectrometer. High-resolution instruments, including Orbitraps and modern time-of-flight systems, can measure peptide masses to within a few parts per million, while lower-resolution instruments are usually read to within a fraction of a dalton. A good report gives the observed mass, the theoretical mass and the difference, so you can judge for yourself how convincing the match is.
What is tandem mass spectrometry used for?
Tandem mass spectrometry, or MS/MS, isolates a single chosen ion, fragments it, and measures the pieces. In peptides, fragmentation along the backbone gives b and y ion series spaced by individual amino acid residues. Reading those ladders can confirm the sequence and distinguish a peptide from another molecule that just happens to have the same intact mass.
REFScientific references
-
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 -
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 -
Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988;60(20):2299-301. PubMed 3239801
original analytical method paper -
Hu Q, Noll RJ, Li H, et al. The Orbitrap: a new mass spectrometer. J Mass Spectrom. 2005;40(4):430-43. PubMed 15838939
review (instrumentation) -
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
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