Ionization techniques are crucial in proteomics, with ESI and MALDI being the most common. ESI produces multiply charged ions and works well with liquid chromatography, while MALDI generates singly charged ions and is great for solid samples and tissue imaging.

Both techniques preserve molecular structure, but their mechanisms differ. ESI involves nebulization and droplet formation, while MALDI uses laser-induced desorption. Each has unique advantages and sample prep methods, making them valuable tools for protein analysis.

Ionization Techniques in Proteomics

ESI vs MALDI techniques

• ESI produces multiply charged ions through continuous ion production facilitating coupling with liquid chromatography (LC-MS)
• MALDI generates primarily singly charged ions via pulsed ion production enabling analysis of solid samples and tissue imaging
• Sample state differs with ESI using analytes in solution while MALDI co-crystallizes analytes with matrix (sinapinic acid)
• Both considered soft ionization techniques preserving molecular structure of large biomolecules (proteins)

Ion formation mechanisms

• ESI mechanism involves:
  1. Nebulization of analyte solution into charged droplets
  2. Solvent evaporation and droplet shrinkage
  3. Coulomb explosion of droplets when surface charge exceeds Rayleigh limit
  4. Ion formation via evaporation model or charge residue model
• MALDI mechanism proceeds through:
  1. Co-crystallization of analyte with matrix (α-cyano-4-hydroxycinnamic acid)
  2. Laser pulse absorption by matrix molecules (nitrogen laser, 337 nm)
  3. Matrix sublimation and expansion into gas phase
  4. Proton transfer from matrix to analyte
  5. Formation of predominantly singly charged ions

Advantages in proteomics applications

• ESI advantages include compatibility with LC enabling online separation, high sensitivity for large biomolecules (antibodies), and extended mass range due to multiple charging
• ESI limitations encompass ion suppression in complex mixtures and requirement for sample desalting
• MALDI advantages comprise salt tolerance, simple mass spectra interpretation, suitability for imaging mass spectrometry (tissue sections), and high throughput capability
• MALDI limitations involve lower quantitative accuracy compared to ESI and matrix interference in low mass range ($<$500 Da)

Sample preparation methods

• ESI sample preparation requires:
  1. Dissolving analyte in suitable solvent (water/acetonitrile mixture)
  2. Adding organic modifier to enhance ionization (methanol)
  3. Adjusting pH for optimal charge state distribution (formic acid)
  4. Desalting using solid-phase extraction or dialysis
  5. Adjusting concentration for stable electrospray ($~$1-10 µM)
• MALDI sample preparation involves:
  1. Selecting appropriate matrix (sinapinic acid for proteins)
  2. Mixing analyte with matrix solution (1:1 ratio)
  3. Spotting on MALDI plate (stainless steel)
  4. Drying to form co-crystals
  5. Optional washing steps to remove salts (ammonium bicarbonate)