Electrospray Ionization

Question 1

Electrospray Ionization (4)

Answer 1

• soft ionization methods
• can be operated in positive or negative modes (i.e. positive mode is for positive gas-phase ions)
• sample can be prepared in an acidic buffer with an organic solvent (denatured ESI) or in an aqueous buffer with a physcial pH and no organic solvent (native ESI)
• sample is pumped through a capillary column at microliters per minute. the capillary is coated with metal and a high voltage (1-5kV) is applied to the end

Question 2

Electrospray Mechanism (3)

Answer 2

1. Droplet Formation
   
   • electrostatic force in the liquid causes oppositely charged ions to migrate away from one another
   • Coulombic repulsion and surface tension cause a “Taylor Cone” at the tip of the capillary
   • with increasing electric force due to the applied voltage charged droplets micormeters in size can break away
   • when operated in the positive mode oxidation in the solution occurs at the metal contact and reduction at the counterelectrode
2. Droplet Shrinkage
   
   • evaporation leads to smaller charged droplets
     
     • charge density of the droplets increases as they shrink leading to the Rayleigh instability limit
   • repulsive Coulombic forces exceed surface tension causing fission of droplets into smaller and highly charged offspring droplets
3. Desorption of Gaseous Ions
   
   • as solvent evapotation and droplet fission is repeated several times a nanometer sized highly charged droplet remains
   • depending upon how the ESI was conducted gas phase positive ions are generated by a specific mechanism

Question 3

Initial Droplet Charge

Answer 3

• protons are often the main contribututor to net droplet charge
  
  • they can come from acidic buffer and can also be generated at the metal/solution interface inside the capillary (the ESI represents an electrochemical cell)

Question 4

Gas Phase Ion Generation

Ion Evaporation Model

Answer 4

• ejection of low molecular weigh species into the gas phase

Question 5

Gas Phase Ion Generation

Charged Residue Model

Answer 5

• native ESI technique
• large globular species such as natively folded proteins are released into the gas phase
• proteins are prepared in an aqueous buffer (ammonium acetate or ammonium bicarbonate) with a pH close to 7 (physical pH) with no organics
• important for the analysis of protein-protein interactinoos or protein-small molecular interactions
• it can keep the non-covalent interaction and allows for the topological investigation of intact protein complexes

Question 6

Gas Phase Ion Generation

Chain Ejection Model

Answer 6

• disordered, partially hydrophobic, and capable of binding excess charge carriers
• unfolded proteins/peptides
• denatured ESI technique
• proteins are dissolved in acidic buffers with organic solvents

Question 7

Native ESI

Answer 7

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