ESI-MS (Electrospray Ionization)

ESI (Electrospray Ionization) is the second major soft ionization technique used in biomolecular mass spectrometry, developed by John Fenn (2002 Nobel Prize shared with Tanaka). It has become the reference method when coupling ionization to liquid chromatography and analyzing continuous-flow samples.

Principle: the solution sample (water-methanol or water-acetonitrile with 0.1 % formic acid or TFA) is infused at low flow rate (1-1000 μL/min) through a capillary held at high potential (±2 to ±5 kV) relative to the counter-electrode. The electric field forms a Taylor cone at the capillary tip, then a nebulized spray of fine charged droplets. Under drying gas flow (nitrogen) and a thermal gradient, droplets evaporate solvent, split (coulombic fissions), and ultimately release gas-phase molecular ions.

The distinctive feature of ESI is its ability to generate multi-charged ions for peptides and proteins of significant size. A 5 kDa peptide can appear as [M+3H]³⁺, [M+4H]⁴⁺, [M+5H]⁵⁺ ions, etc. This multi-charging shifts peaks to lower m/z values compatible with most analyzers' range and allows analysis of proteins of several tens of kDa on classical instruments.

LC-MS (liquid chromatography mass spectrometry) coupling is the flagship application. An acetonitrile/water gradient separates peptides of a tryptic digest on reverse-phase C18, and flow goes directly into the ESI source. High-resolution mass spectrometry (Orbitrap, QTOF, FT-ICR) then provides exact mass (1-5 ppm accuracy) and MS/MS fragmentation for sequence identification.

Quantitative analysis benefits from techniques like SRM/MRM (Selected/Multiple Reaction Monitoring) on triple quadrupoles: specific precursor ion-fragment ion pairs allowing absolute quantification at ng/mL in complex biological matrices (plasma, urine, tissue homogenate). Stable isotope internal standards (²H, ¹³C, ¹⁵N) give 5-10 % precision acceptable in pharmacokinetics.

ESI strengths: LC coupling for complex mixtures, multi-charging for large molecules, robust quantification, compatible with all modern analyzer types. Limitations: sensitivity to salts and detergents (< 50 mM NaCl usually), need for compatible volatile solvent, ion suppression in biological matrices, more complex spectra to interpret for novices than MALDI.