7.5 Protein analysis using electrospray ionization mass spectroscopy

Introduction

Electrospray ionization-mass spectrometry (ESI-MS) is an analytical method that focuses on macromolecular structural determination. The unique component of ESI-MS is the electrospray ionization. The development of electrospraying, the process of charging a liquid into a fine aerosol, was completed in the 1960’s when Malcolm Dole ( [link] ) demonstrated the ability of chemical species to be separated through electrospray techniques. With this important turn of events, the combination of ESI and MS was feasible and was later developed by John B. Fenn ( [link] ), as a functional analytical method that could provide beneficial information about the structure and size of a protein. Fenn shared the Nobel Prize in 2002, with Koichi Tanaka ( [link] ) and Kurt Wüthrich ( [link] ) for the development of ESI-MS.

ESI-MS is the process through which proteins, or macromolecules, in the liquid phase are charged and fragmented into smaller aerosol droplets. These aerosol droplets lose their solvent and propel the charged fragments into the gas phase in several components that vary by charge. These components can then be detected by a mass spectrometer. The recent boom and development of ESI-MS is attributed to its benefits in characterizing and analyzing macromolecules, specifically biologically important macromolecules such as proteins.

How does esi-ms function?

ESI-MS is a process that requires the sample to be in liquid solution, so that tiny droplets may be ionized and analyzed individually by a mass spectrometer. The following delineates the processes that occur as relevant to [link] :

• Spray needle/capillary- The liquid solution of the desired macromolecule is introduced into the system through this needle. The needle is highly charged via an outside voltage source that maintains the charge constant across the needle. The normal charge for a needle is approximately 2.5 to 4 kV. The voltage causes the large droplets to fragment into small droplets based on charge that is accumulated from the protein constituent parts, and the liquid is now in the gas phase.
• Droplet formation- The droplets that are expelled from the needle are smaller than initially, and as a result the solvent will evaporate. The smaller droplets then start increasing their charge density on the surface as the volume decreases. As the droplets near the Rayleigh limit, Coulombic interactions of the droplet equal the surface tension of the droplet, a Coulombic explosion occurs that further breaks the droplet into minute fractions, including the isolated analyte with charge.
• Vacuum interface/cone - This portion of the device allows for the droplets to align in a small trail and pass through to the mass spectrometer. Alignment occurs because of the similarity and differences in charges amongst all the droplets. All the droplets are ionized to positive charges through addition of protons to varying basic sites on the droplets, yet all the charges vary in magnitude dependent upon the number of basic sites available for protonation. The receiving end or the cone has the opposite charge of the spray needle, causing an attraction between the cone and the droplets.
• Mass spectrometer- The charged particles then reach the mass spectrometer and are deflected based on the charge of each particle. Deflection occurs by the quadrupole magnet of the mass spectrometer. The different deflection paths of the ions occur due to the strength of the interaction with the magnetic field. This leads to various paths based on a mass/charge ( m/z ) ratio. The particles are then read by the ion detector, as they arrive, providing a spectrum based on m/z ratio.