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Mass spectrometry using electrospray ionization

Nature Reviews Methods Primers volume 3, Article number: 23 (2023) Cite this article

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Abstract

Applying a high voltage to a liquid passing through a capillary disintegrates the liquid meniscus to generate a fine spray of charged droplets. As the droplets evaporate, polar molecules dissolved in the liquid are ionized and brought into the gas phase. This phenomenon is commonly known as electrospray ionization (ESI). The gaseous ions can be analysed using mass spectrometry (MS) to determine their molecular masses and chemical structures. Polar analytes — including low-molecular-weight species such as amino acids, peptides, nucleotides and synthetic chemicals; macromolecules, for example, synthetic and natural polymers; and nanoscale particles, for instance, viruses — are ionizable using ESI. In the past three decades, ESI-MS has evolved into a powerful instrumental approach useful to (bio)chemists in a wide range of applications. Several different variants have been developed to extend its functionality. For obtaining chemical and structural information on macromolecules, ESI-MS is complementary to fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, X-ray crystallography and cryo-electron microscopy. This Primer discusses the ESI setup, mechanism and variants. It further introduces sample and instrument-related factors that affect the ionization process, applications and current research, as well as giving a future perspective.

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Fig. 1: A typical electrospray ionization setup.
Fig. 2: High-speed imaging of the disintegration of a levitated droplet charged to the Rayleigh’s limit.
Fig. 3: Electrospray ionization-mass spectrometry system.
Fig. 4: Graphic illustrating main variants of electrospray ionization.
Fig. 5: Matrix effect observed in electrospray ionization-mass spectrometry.
Fig. 6: Representative electrospray ionization mass spectra of different compounds acquired using low-resolution and high-resolution mass spectrometers.
Fig. 7: Proteomic analysis.

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Acknowledgements

P.L.U. acknowledges the National Science and Technology Council, Taiwan (grant numbers 110-2628-M-007-004-MY4, 109-2113-M-007-013-MY3, 111-2634-F-007-007 and 110-2811-M-007-513), the National Tsing Hua University (111QI009E1), the Frontier Research Center on Fundamental and Applied Sciences of Matters as well as the Featured Areas Research Center Programme within the framework of the Higher Education Sprout Project established by the Ministry of Education, Taiwan (111QR001I5).

Author information

Authors and Affiliations

  1. Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan

    Gurpur Rakesh D. Prabhu & Pawel L. Urban

  2. Department of Chemistry, University of California, Berkeley, CA, USA

    Evan R. Williams

  3. Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland

    Matthias Wilm

  4. Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu, Taiwan

    Pawel L. Urban

Authors
  1. Gurpur Rakesh D. Prabhu
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  2. Evan R. Williams
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  3. Matthias Wilm
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  4. Pawel L. Urban
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Contributions

Introduction (M.W. and G.R.D.P.); Experimentation (G.R.D.P., M.W., E.R.W. and P.L.U.); Results (M.W. and G.R.D.P.); Applications (G.R.D.P., E.R.W. and P.L.U.); Reproducibility and data deposition (P.L.U. and G.R.D.P.); Limitations and optimizations (G.R.D.P. and P.L.U.); Outlook (P.L.U. and G.R.D.P.) and Overview of the Primer (G.R.D.P., M.W., E.R.W. and P.L.U.).

Corresponding author

Correspondence to Pawel L. Urban.

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The authors declare no competing interests.

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Nature Reviews Methods Primers thanks the anonymous reviewers for their contribution to the peer review of this work.

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Related links

Human Metabolome Database: https://hmdb.ca/

MASH explorer: https://labs.wisc.edu/gelab/MASH_Explorer/index.php

MassBank: http://www.massbank.jp/

MassBank-EU: https://massbank.eu/MassBank/

MassIVE: https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp

Mass Spectrometry Data Center: https://chemdata.nist.gov/dokuwiki/doku.php?id=start

MetaMorpheus: https://smith-chem-wisc.github.io/MetaMorpheus/

Metlin: https://massconsortium.com/

MoNA: https://mona.fiehnlab.ucdavis.edu/

MZmine: http://mzmine.github.io/

PeakInvestigator: https://www.veritomyx.com/PeakInvestigator.php

PRIDE: https://www.ebi.ac.uk/pride/

ProMass: https://www.enovatia.com/our-products/promass/

PubChem: https://pubchem.ncbi.nlm.nih.gov/

UniDec: http://unidec.chem.ox.ac.uk/

Supplementary information

Glossary

Charged residue model

A model to explain the ionization mechanism for electrospray droplets containing a single analyte, such as a globular protein, in which the charge present on the droplet surface is transferred to the analyte, as the solvent shell evaporates to dryness.

Collision cell

A zone inside a mass spectrometer in which gas-phase ions are accelerated and dissociated into fragments by energetically colliding the ions with neutral particles — for example, gaseous atoms or molecules (helium, argon, nitrogen) — or by interacting with electrons, or by absorbing photons (infrared or ultraviolet).

Coulombic jet fission

The process of ejecting small charged droplets from larger charged droplets owing to prevalence of electrostatic repulsion force over surface tension force.

Desolvation

The process of solvent evaporation from electrospray droplets and formation of dry ions.

Ionization chamber

A region at the front end of a mass spectrometer in which the ion source is located.

Ion suppression

Decrease of ionization efficiency of target analytes owing to the presence of interfering species in the sample.

Lorentz force

A force exerted on an ion while it is moving through an electric field and a magnetic field.

Multiply-charged ion

An ion with charge number greater than 1. Macromolecules form multiply-charged ions during the electrospray ionization process.

m/z value

The mass-to-charge ratio of an analyte measured using a mass spectrometer.

Nano-electrospray

Electrospray generated using a thin emitter, with a tip inner diameter in the order of a few micrometres, to produce a fine spray of primary droplets with size in the range of a few hundred nanometres.

Plume

Cloud of charged droplets projecting from Taylor cone.

Progeny droplets

A set of nanometre-sized secondary droplets produced during fission of the primary micrometre-sized electrospray droplets.

Resolving power

The ability of a mass analyser to distinguish two closely spaced peaks of different m/z values in a mass spectrum.

Taylor cone

Elongated liquid meniscus at the electrospray capillary outlet.

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Prabhu, G.R.D., Williams, E.R., Wilm, M. et al. Mass spectrometry using electrospray ionization. Nat Rev Methods Primers 3, 23 (2023). https://doi.org/10.1038/s43586-023-00203-4

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