Protein ionization analytical methods

Electrospray ionization mass spectrometry (ESI-MS) is an analytical method for mass determination of ionized molecules. It is a commonly used method for soft ionization of peptides and proteins in quadmpole, ion-trap, or time-of-flight mass spectrometers. The ionization is performed by application of a high voltage to a stream of liquid emitted from a capillaty. The highly charged droplets are shrunk and the resulting peptide or protein ions are sampled and separated by the mass spectrometer. 

FIGURE 15.2 Common protein ionization methods used for MS-based proteomics. Two common ionization technologies are currently available for protein analysis. Top ESI volatilizes and ionizes peptides and proteins in solution. Bottom MALDI uses analytes that are co-crystallized in a matrix composed of organic acid on a solid support. A pulse of ultraviolet laser evaporates the matrix and analyte into gas phase, resulting in generation of single charge ions. 

Unlike nucleic acids, proteins in an organism are present at very different concentration levels. Thus, it is not sufficient to demonstrate that a particular protein is present we also need to know its concentration. From the high-concentration globulins in blood to the low-copy-number proteins that are represented by only a few molecules per cell, there is an enormous dynamic range. This presents a challenge to the utilized analytical methods because of the potential interferences, especially when quantitating the proteins at low concentration. For example, the high-abundance proteins can compete in the ionization process and suppress the ion formation from the low-level species. This ion suppression effect is quite common in MALDI and ESI ion sources. 

Electrospray ionization (ESI) is a method by which solutes present in a solution can be transferred into the gas phase as ions. The gas-phase ions can then be detected by mass spectrometric means (ESIMS). Remarkably, ESI can handle solutes such as polymers, nucleic acids, and proteins that have a very high molecular mass such as hundreds of megadaltons for proteins. The analytes present in the solution may be ions, such as the inorganic metal ions M and protonated amines or negative ions such as the halide ions... 

ESI has become the most commonly used interface for LC/MS. It was recognized by John Fenn and co-workers as an important interface for LC/MS immediately after they developed it as an ionization technique for MS. ESI transforms ions in solution to ions in the gas phase and may be used to analyze any polar molecule that makes a preformed ion in solution. The technique has facilitated the ionization of heat-labile compounds and high-molecular-weight molecules such as proteins and peptides. ESI is a continuous ionization method that is particularly suitable for use as an interface with FiPLC. It is the most widely accepted soft-ionization technique for the determination of molecular weights of a wide variety of analytes and, has made a significant impact on drug discovery and development since the late 1980s. 

Electrospray (ESI) is an atmospheric pressure ionization source in which the sample is ionized at an ambient pressure and then transferred into the MS. It was first developed by John Fenn in the late 1980s [1] and rapidly became one of the most widely used ionization techniques in mass spectrometry due to its high sensitivity and versatility. It is a soft ionization technique for analytes present in solution therefore, it can easily be coupled with separation methods such as LC and capillary electrophoresis (CE). The development of ESI has a wide field of applications, from small polar molecules to high molecular weight compounds such as protein and nucleotides. In 2002, the Nobel Prize was awarded to John Fenn following his studies on electrospray, for the development of soft desorption ionization methods for mass spectrometric analyses of biological macromolecules. ... 

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