Electrospray ionization: “We taught elephants to fly” Open Access

The concept of electrospray ionization (ESI) was first coupled with mass spectrometry (MS) in the late 1960s by Malcom Dole as a technique to ionize, and subsequently analyse, high molecular weight synthetic polymers. It wasn’t until 20 years later, however, that ESI was used to ionize biologically important ‘molecular elephants’, i.e., high molecular weight biopolymers, such as proteins and resultant peptides. Here, John Fenn pioneered the ‘soft ionization’ of proteins from solution-phase analytes into gas-phase ions for analysis by MS. These experiments permitted, for the first time, the global and integrated study of the proteome, allowing the identification of protein biomarkers for early disease diagnosis, prognosis and development of therapeutic strategies. For this work, John Fenn was awarded a share of the 2002 Nobel Prize in chemistry for ‘development of methods for identification and structure analysis of biological macromolecules’. Fenn shared the prize with Koichi Tanaka and Kurt Wüthrich for their work on MALDI and NMR, respectively.

ESI is a ‘soft ionization’ technique used to create gaseous ions from large highly polar compounds (i.e., proteins) contained in liquid solvents by multiple charging. ‘Soft ionization’ is an intrinsic and important feature of ESI. Prior to its development in 1989, high molecular weight biopolymers, such as proteins and their resultant peptides, were unable to be ionized without undergoing extensive and detrimental fragmentation. The unique ‘soft’ nature of ESI means very little fragmentation occurs during the ionization process, allowing weak, non-covalent interactions to be maintained in the gas phase. This is particularly advantageous when analysing high molecular weight biopolymers, such as proteins and peptides as they contain a host of noncovalent interactions. Multiple charging is also an important feature of ESI, especially when analysing high molecular weight biopolymers. ESI is able to produce multiple charged ions, dependent/proportional to their molecular mass. A crucial consequence is that the mass-to-charge (or m/z) values of the resultant protein or peptide ions are reduced, and fall within the mass range for MS (typically 300–2000 m/z).

One of the key advantages of ESI is its ability to take analytes in the liquid phase, for example from high-performance liquid chromatography (LC) systems, or capillary electrophoresis, and produce gas-phase ions. The mechanism of ESI, using proteomic analysis of peptides as an example, takes place at the interface between the LC system and the ion source region of the mass spectrometer (Figure 1). Here, solution-phase analytes are converted into gas-phase ions in three stages: aerosol formation, solvent evaporation (or desolvation) and gaseous ion formation.

In the first step, an analyte solution, i.e., peptide mixture contained in a volatile liquid effluent, is infused into a metal capillary close to the inlet of the mass spectrometer. As the analyte solution reaches the end of the column, it flows through a fine spray needle. Here, peptide ions are generated by applying a high electric voltage to the liquid effluent, relative to the inlet of the mass spectrometer, thus producing an aerosol in which the peptides become contained in highly positively charged droplets (Figure 2).

Next, the solvent continuously evaporates (also termed desolvation), with the help of a heated dry gas counter-current (i.e., nitrogen), from the positively charged droplets resulting in an increasing size-to-charge ratio until the Rayleigh limit is reached, i.e., the point at which the size-to-charge ratio of the liquid droplet becomes so great that it throws out fine jets of liquid – electrospray (Figure 2).

Finally, the size of the charged droplets becomes so small, reaching the nanometre level; the peptide ions contained within the liquid droplets turn into gas-phase ions, with several models existing to explain this process (Figure 2).

A peptide mixture contained in a volatile liquid effluent is loaded onto a high-performance liquid chromatography (HPLC) column. A high voltage is applied to the tip of the column (spray needle), relative to the inlet of the mass spectrometer, which causes the dispersion of the sample solution into an aerosol of highly charged electrospray droplets.

In a typical ESI-LC-MS proteomic workflow, ionization is largely achieved by performing ESI in positive (ESI+) mode, where charging occurs via protonation ([M + H]⁺, the addition of a proton) under acidic conditions. Protonation most typically occurs on the most basic site of the protein, i.e., those containing an amine functional group. Multiple charged ions occur, therefore, when more than one basic site is available. The efficiency of this ionization process varies from protein to protein and is highly dependent on solvent, pH and other instrumental parameters such as spray voltage, source gas pressure and heat.

The uses of ESI are by no means limited to proteomics, but in this article we primarily use examples from this field. Proteomics is defined as the large-scale characterization of the entire protein complement, i.e., the proteome, of a cell, tissue, organism or biofluid with regard to protein expression, localization, interactions, post-translational modifications (PTMs) and turnover. The proteome adds huge complexity to the genome, where protein levels are dependent not only on mRNA levels but also on a host of translational control mechanisms and regulation that creates a highly complex proteome which exhibits an extensive dynamic range of at least 12 orders of magnitude.

The use of ESI-MS in proteomic research has facilitated the accurate and timely measure of thousands of proteins in complex biological matrices with sub-femtomole (fmol, 10⁻¹⁵ mol) sensitivity. In this application, MS is used to measure, and quantify, the m/z of ionized biomolecules, either proteins or their resultant peptides, providing information on sample composition, PTMs and protein structure. In this technique, proteins can be studied either as intact entities by top-down MS methods or as peptides produced by the enzymatic digestion of proteins by bottom-up MS methods. Bottom-up methods, unlike top-down, provide a global insight into the proteome, enabling the detection of thousands of proteins in a single biological sample. Bottom-up proteomic methods using ESI are now the standard method for analysing PTMs high throughput, with kit-based commercial solutions available for preparing samples enriched in a subset of PTMs such as phosphopeptides, or ubiquitin-modified peptides. Published methods further expand the range of PTMs available for analysis, though the study of acid-labile modifications is possible but challenging for analysis under the standard acidic LC conditions typically used for peptide analysis. Lastly, using ESI, intact proteins can be analysed, with the ‘soft’ ionization afforded by ESI enabling the analysis of protein complexes or even complexes of proteins and other molecules such as nucleic acids. LC buffer conditions can be adjusted for analysis of proteins under native or denatured conditions.

Underpinned by ESI, significant advances in proteomic methods have arisen in the past decades that have permitted the global and integrated study of the proteome, allowing the identification of protein biomarkers for early disease diagnosis, prognosis and development of therapeutic strategies. Even today, emerging applications in mass spectrometry-based proteomics are underpinned by ESI. Our lab works on one such area – single-cell proteomics by mass spectrometry, where individual mammalian cells are trypsin digested, labelled with isobaric tagging reagents and analysed by LC-MS/MS in small batches of 14 single cells (SCoPE2). Other emerging approaches in proteomics include single-molecule applications and structural analysis. The dominance of ESI-based methods in mass spectrometry-based proteomics research appears set to continue well into the future.

• The original research article from Malcom Dole’s lab: Malcolm Dole, L. L. Mack, and R. L. Hines (1968) "Molecular beams of macroions", J. Chem. Phys. 49, 2240–2249. https://doi.org/10.1063/1.1670391

• The original research article on ESI from John Fenn’s lab: Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., & Whitehouse, C. M. (1989). “Electrospray ionization for mass spectrometry of large biomolecules”, Science, 246(4926), 64–71. https://doi.org/10.1126/science.2675315

• Comprehensive review of electrospray ionisation and its application development: Cech, N. B., & Enke, C. G. (2001). “Practical implications of some recent studies in electrospray ionization fundamentals”, Mass Spectrometry Reviews, 20(6), 362–387. https://doi.org/10.1002/mas.10008

• A recent review highlighting the uses of mass-spectrometry based proteomics: Aebersold, R., Mann, M. (2016). “Mass-spectrometric exploration of proteome structure and function”, Nature 537, 347–355. https://doi.org/10.1038/nature19949

Rosemary Maher graduated with a BSc in biomedical Science from the University of Sheffield in 2016 and has recently received her PhD from the University of Liverpool in 2022. During her PhD, Rosemary used mass spectrometry-based proteomic methods to assess respiratory disease and pulmonary aspiration, in patients with cystic fibrosis and severe neurodisability and in dogs, idiopathic pulmonary fibrosis. The overall goal of Rosemary’s PhD was to provide an understanding of the molecular mechanisms that underpin respiratory disease in these clinical cohorts by highlighting proteins and protein pathways involved in disease pathogenesis and lung function decline. Rosemary now holds a postdoctoral position within the Centre for Proteome Research (Emmott Lab) at the University of Liverpool continuing her research into cystic fibrosis lung disease, specifically investigating elexacaftor–tezacaftor–ivacaftor, a novel CFTR modulator treatment that produces dramatic clinical improvements in cystic fibrosis patients, and its effect on the sputum and plasma proteome. Twitter: @rosiemaher Email: [email protected].

Edward Emmott graduated with a BSc in medical microbiology and virology from the University of Warwick in 2007 and a PhD from the University of Leeds in 2011. He held postdoctoral positions at Imperial College London, the University of Cambridge and the Northeastern University studying virus–host interactions and multi-component protein complexes and developing new methods for single-cell proteomics. He joined the Centre for Proteome Research at the University of Liverpool in 2019 as a Tenure-Track Fellow. His research group uses and develops mass spectrometry approaches to study virus replication and virus–host interactions, as well as in clinical collaborations. Twitter: @edemmott/@emmottlab Email: [email protected].