Mass Spectrometer
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MS training course Vikas Kumar, PhD "In recognition of the great merits of his theoretical and experimental investigations on the conduction of electricity by gases.“ 1906 Nobel Prize Cited from: http://masspec.scripps.edu/mshistory/ First MS called “Parabola spectrograph” Ion were separated by different parabolic trajectories in electromagnetic field. Detection by the ions striking a fluorescent screen or photographic plate. Designed a mass spectrometer in which ions were dispersed by mass and focused by velocity--which improved MS resolving power by an order of magnitude over the resolution Thomson had been able to achieve. Aston’s design was the basis of his later instruments with which he systematically and accurately measured the masses of the isotopes of many of the elements. Francis William Aston 1922 Nobel Prize "For his discovery, by means of his mass spectrograph, of isotopes, in a large number of non-radioactive elements, and for his enunciation of the whole-number rule." Cited from: http://masspec.scripps.edu/mshistory/ In 1920 A. J. Dempster developed single focusing magnetic deflection instrument. He also developed the first electron impact source, which ionizes volatilized molecules with a beam of electrons from a hot wire filament. In 1940s Alfred O. C. Nier developed sector field MS. It was used during World War II to do isotopic analysis, with separation of uranium-235 from uranium-238. The Calutron, a three-story-high version of Nier's magnetic sector instrument, separated uranium-235 for the first atomic bomb. Cited from: http://masspec.scripps.edu/mshistory/ In 1946 W. E. Stephen from University of Pennsylvania proposed TOF in that ions are separated by differences in their velocities as they move in a straight path toward a collector in order of increasing mass-to-charge ratio. Improved by W. C. Wiley and I. H. McLaren from Bendix Corp., Detroit, Mich in mid 1950s and further improved by invention of reflectron B. A. Mamyrin in 1974. When commercial TOF instruments first came out their performance in resolution was so poor that they never lived up to even single-focusing magnetic instruments. However, this analyzer has been greatly improved recently to almost match the most sophisticated, and very expensive Ion cyclotron resonance MS. Cited from: http://masspec.scripps.edu/mshistory/ Quandrupole was first reported in the mid-1950s by Wolfgang Paul of the University of Bonn. Then Paul also developed quadrupole ion Wolfgang Paul trap, which can trap and mass-analyze ions using a three-dimensional quadrupolar radiofrequency electric field. An ion trap system was first introduced Hans Georg Dehmelt commercially in 1983 by Finnigan MAT. “For the development of the ion trap technique.” 1989 Nobel prize Cited from: http://masspec.scripps.edu/mshistory/ 1956 Gas Chromatography Mass Spectrometry (GC/MS) 1956 Identifying Organic Compounds with Mass Spectrometry 1962 Mass Spectrometry Imaging 1966 Chemical Ionization 1966 Peptide Sequencing 1966 Tandem Mass Spectrometry 1966 Metabolomics 1968 Electrospray Ionization 1968 Collision Induced Dissociation Cited from: http://masspec.scripps.edu/mshistory/ 1974 Fourier Transform Ion Cyclotron Resonance 1974 Extra-Terrestrial Mass Spectrometry 1975 Atmospheric Pressure Chemical Ionization (APCI) 1978 Triple Quadrupole Mass Analyzer 1980 Inductively Coupled Plasma MS 1984 Quadrupole/Time-Of-Flight Mass Analyzer 1987 Soft Laser Desorption of Proteins 1989 Monitoring Enzyme Reactions with ESI-MS Cited from: http://masspec.scripps.edu/mshistory/ 1990 Protein Conformational Changes with ESI-MS 1990 Clinical Mass Spectrometry 1991 MALDI Post-Source Decay 1991 Non-covalent Interactions with ESI 1992 Low Level Peptide Analysis 1993 Oligonucleotide Ladder Sequencing 1993 Protein Mass Mapping 1996 Intact Virus Analyses Cited from: http://masspec.scripps.edu/mshistory/ Two recently developed MS techniques have had a major impact on the ability to use MS for the study of large biomolecules: electrospray ionization MS (ESI MS) and ESI matrix-assisted laser desorption/ionization MS (MALDI MS). ESI was first conceived in the 1960s by Malcolm Dole of John B. Fenn Northwestern University, Evanston, but it was put into practice in the early 1980s by John B. Fenn of Yale University. MALDI MS, a form of laser desorption MS, was developed in MALDI 1985 at the University of Frankfurt, Germany, by Franz Hillenkamp, and independently by Koichi Tanaka and coworkers at Shimadzu Corp., Kyoto, Japan. Koichi Tanaka "For the development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules.“ 2002 Nobel prize Cited from: http://masspec.scripps.edu/mshistory/ 1998 Electron Capture Dissociation (ECD) 1999 Quantitative Proteomics and Metabolomics with Isotope Labels 2000 Orbitrap 2004 Electron Transfer Dissociation (ETD) 2005 Direct Analysis in Real Time (DART) 2014 Draft of Human Proteome Cited from: http://masspec.scripps.edu/mshistory/ Mass spectrometry is a powerful analytical technique that is used to Identify unknown compounds. Quantify known compounds. Elucidate the structure and chemical properties of molecules. Highly sensitive, It can detect compounds in very minute quantities (femtomoles). Mass Spectrometer (Smallest scale) A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted to ions; i.e., molecules that have been electrically charged. Ionization Mass Sorting (filtering) Detection Ion Ion Source Mass Analyzer Detector Form ions Sort Ions by Mass (m/z) (charged molecules) Detect ions 100 75 Inlet • Solid 50 • Liquid 25 0 • Vapor 1330 1340 1350 Mass Spectrum Atmospheric Low Pressure (~10-6 torr) Pressure (760 torr) Direct Insertion: Done using insertion plate/probe. The sample is placed onto a probe having loading chamber separated from ionization region of the mass spectrometer through a vacuum interlock. Direct Infusion: A simple capillary or a capillary column is used to introduce a sample as a gas or in solution. Direct infusion is also useful because it can efficiently introduce small quantities of sample into a mass spectrometer without compromising the vacuum. Capillary columns are routinely used to interface separation techniques with the ionization source of a mass spectrometer. Protonation: In this method proton is added to a molecule, producing a net positive charge of 1+ for every proton added. M + H+ MH+ Positive charges tend to reside on the more basic residues of the molecule, such as amines, to form stable cations. MALDI, ESI and APCI use this approach. Some compounds are not stable to protonation (i.e. carbohydrates) or cannot accept a proton easily (i.e. hydrocarbons). Deprotonation: in this method net negative charge is achieved through the removal of a proton from a molecule. M - H+ M-H- This mechanism of ionization, commonly achieved via MALDI, ESI, and APCI is very useful for acidic species including phenols, carboxylic acids, and sulfonic acids. This method is compound specific. Cationization: in this positively charged ion is produced by non-covalently adding a positively charged ion to a neutral molecule. M + C+ MC+ Although similar to protonation however it refers to addition of a cation adduct other than a proton (e.g. alkali, ammonium). Cationization is commonly achieved via MALDI, ESI, and APCI. Carbohydrates are excellent candidates for this ionization mechanism, with Na+ a common cation adduct. Limited information in MS/MS Electron ejection: In this method ionization through the ejection of an electron to produce a 1+ net positive charge, often forming radical cations. M -e- M+ Observed most commonly with electron ionization (EI) sources, electron ejection is usually performed on relatively nonpolar compounds with low molecular weights and it is also known to generate significant fragment ions. Often generates too much fragmentation, it can be unclear whether the highest mass ion is the molecular ion or a fragment. Electron capture: In this method a net negative charge of 1- is achieved with the absorption or capture of an electron. M +e- M- It is a mechanism of ionization primarily observed for molecules with a high electron affinity, such as halogenated compounds. Often generates too much fragmentation, it can be unclear whether the highest mass ion is the molecular ion or a fragment. Ionization Source Acronym Event Electrospray ionization ESI evaporation of charged droplets Nanoelectrospray ionization nanoESI evaporation of charged droplets Atmospheric pressure chemical ionization APCI corona discharge and proton transfer Matrix- assisted laser desorption/ionization MALDI photon absorption/proton transfer Fast atom/ion bombardment FAB ion desorption/proton transfer Electron ionization EI electron beam/electron transfer Chemical ionization CI proton transfer • A mass spectrometer does not measure mass, it measures the mass-to-charge ratio (m/z), z must not be zero. • Ionization can be achieved by adding or removing a charged particle. • A mass spectrum is an intensity vs. m/z plot representing a chemical analysis. • Peak intensity is NOT a linear response to the abundance because of the differences in ionization and detection efficiencies. It is also energy dependent. The most intense peak is called the base peak and is arbitrarily assigned the relative abundance of 100 %. F: FTMS + c NSI Full ms [300.00-1000.00] 421.76 100 90 80 450.25 70 60 50 529.81 40 674.86 Relative Abundance Relative 30 540.80 353.54 20 523.29 842.51 10 652.85 310.83 396.24 465.74