Gradually a systematic analysis of proteins with the mass spectrometry as the central tool led to a discrete subject called “Proteomics,” one of the fastest growing research areas in the chemical sciences. Thus ESI became very useful in the production of gas-phase ions from large biologically important macromolecules like proteins and nucleic acids, and their subsequent mass spectrometric analysis for structural characterization as well as their rapid identification on the basis of molecular mass, a very specific property of the analyte. Because of the multiple charging, the m/z values of the resulting ions become lower and fall in the mass ranges of all common mass analyzers. Not only that but also very weak noncovalent interactions are preserved in the gas phase. The ionization is soft in the sense that a very little residual energy is retained by the analyte, and generally no fragmentation occurs upon ionization. So the only way to analyze the protein mass was to digest the protein and then the analysis of the digest mixture by FAB-mass spectrometry.Īll those problems were overcome in 1989 when Fenn introduced electrospray ionization, a soft ionization technique, to ionize intact chemical species (proteins) by multiple charging. However, the available mass analyzers could not measure the high m/z value of the singly charged high molecular weight proteins during those days. Although a technique called fast atom bombardment (FAB) was available that time for the ionization of the biological samples, this technique produces predominantly singly charged ions of the analyte and the method works best for smaller species of mass below about 1000 Da. So the ionization of the proteins by conventional ionization methods could lead to structural destruction. But the proteins are polar, nonvolatile, and thermally labile molecules. In the mid 1980s, it became indispensable to precisely measure the molecular mass of the biologically important supramolecules like proteins. Before the development of ESI-MS, there were several ionization methods (electron ionization, chemical ionization, etc.), but none of them could be able to overcome the propensity of the analyte fragmentation. As a result the mass spectrometry has become one of the most sensitive analytical methods for the structural characterization of molecules. The success of the study of gas-phase ion chemistry and its application has been driven by the continuous advancement of the mass spectrometric technique since the studies were performed by Thomson. In mass spectrometry, a particular state of matter called gaseous ionic state is studied by transferring the analytes from condensed phase to the gas phase followed by their ionization. Since then the analytical measurement of masses of the samples continuously evolved through the gravimetric analysis to weighing a single atom/molecule using the modern instrument called mass spectrometer. The basic concepts of chemistry originated from the quantitative estimation (e.g., weighing) of the constituents in a chemical reaction during the period of Lavoisier more than 200 years ago. The review will highlight recent developments and emerging directions in this fascinating area of research. The charged analytes produced by ESI can be fragmented by activating them in the gas-phase, and thus tandem mass spectrometry has been developed, which provides very important insights on the structural properties of the molecule. Several groups have investigated the origin and implications of the multiple charge states of proteins observed in the ESI-mass spectra of the proteins. There have been extensive studies on the mechanism of formation of charged gaseous species by the ESI. In the present review we have described the development of Electrospray Ionization mass spectrometry (ESI-MS) during the last 25 years in the study of various properties of different types of biological molecules. The Electrospray Ionization (ESI) is a soft ionization technique extensively used for production of gas phase ions (without fragmentation) of thermally labile large supramolecules.
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