Learning Objectives for Mass Spectrometry Lecture.by Dr. Ilya Ioshikhes,Department of Biomedical Informatics, 3017 Graves Hall,Tel. 292-6514, E-mail: [email protected] spectrometry – basic principles and common applications.1. What is mass spectrometry?2. What is the major role of mass spectrometry?3. Bring some examples of applications of mass spectrometry.4. How did mass spectrometry originate?5. What is a mass spectrometer?6. Describe major components of a mass spectrometer.7. What are the characteristics of the mass spectrum?8. What is a mass-to-charge ratio?9. What units are used for it in mass spectrometry?10. What is Resolution of a mass spectrometer?11. How the samples are studied by mass spectrometry?12. What is the analyzer and how it works?13. What is the role of computers in mass spectrometry?14. How can mass spectrometric data be used for structure analysis?15. How large a molecule can be analyzed?16. What other techniques are usually combined with mass spectrometry?17. How is mass spectrometry used for quantitative analysis?Biomedical applications of mass spectrometry.18. What biomedical applications of mass spectrometry do you know?19. What is MALDI-TOF MS?20. How mass spectrometry may be used for determination of a protein mass?21. How mass spectrometry may be used for sequencing of proteins or oligosaccharides?22. How the composition of biological fluids may be revealed by the MALDI-TOF MS?Key information elements.Basic principles of mass spectrometry and its .1. Mass spectrometry: A method used to determine the masses of atoms ormolecules in which an electrical charge is placed on the molecule and theresulting ions are separated by their mass to charge ratio.2. The technique of mass spectrometry had its beginnings in J.J. Thomson's vacuumtube where in the early part of the 20th century the existence of electrons and"positive rays" was demonstrated. The primary application of mass spectrometryremained in physics for nearly thirty years. It was used to discover a number ofisotopes, to determine the relative abundance of the isotopes, and to measure their"exact masses", i.e., atomic masses to within a precision of 1 part in 106 or better.These important fundamental measurements laid the foundation for laterdevelopments in diverse fields ranging from geochronology to biochemicalresearch. 3. Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to elucidate thestructure and chemical properties of molecules. Detection of compounds can beaccomplished with very minute quantities (as little as 10-12g, 10-15 moles for acompound of mass 1000 Daltons). This means that compounds can be identifiedat very low concentrations (one part in 1012) in chemically complex mixtures.Mass spectrometry provides valuable information to a wide range ofprofessionals: physicians, astronomers, and biologists, to name a few.4. A mass spectrometer is an instrument that measures the masses of individualmolecules that have been converted into ions, i.e., molecules that have beenelectrically charged.5. A mass spectrum is a graph of ion intensity as a function of mass-to-charge ratio. In mass spectrometry, Dalton (Da) is used as unit of mass m. 1 Da = (1/12) of the mass of a single atom of the isotope of carbon-12 (12C). Magnitude of charge of anelectron is used as unit of charge z. Mass spectrometry operates with the mass-to-charge ratio m/z in the appropriate units.6. Formation of gas phase samples is an essential beginning step of massspectrometry. Early mass spectrometers required sample to be a gas, laterapplicability of mass spectrometry was extended to liquid and solid samples thatcommonly should be volatilized and ionized.7. The gas phase ions are sorted in the mass analyzer according to their mass-to-charge (m/z) ratios and then collected by a detector. In the detector the ion flux isconverted to a proportional electrical current. The data system records themagnitude of these electrical signals as a function of m/z and converts thisinformation into a mass spectrum.8. Resolution or Resolving Power is the ability of a mass spectrometer to distinguishbetween ions of different mass-to-charge ratios such that greater resolutioncorresponds directly to the increased ability to differentiate ions. For example, amass spectrometer with a resolution of 500 can distinguish between ions of m/z =500 and 501. The most common definition of resolution is given by the followingequation: Resolution = M / ΔM, where M is the m/z of an ion and ΔMis the full-width at half-maximum peak.9. The mass spectrum of a large molecule typically shows many fragment ions inaddition to the molecular ion. The most intense ions have been labeled with theirmass-to-charge ratio. The fragment ions are used for deduction of the molecularstructures.10. Mass spectrometry (MS) is often used in combination with separation techniqueslike gas chromatography (GC), liquid chromatography (LC), capillaryelectrophoresis and supercritical fluid chromatography (SFC).11. Gas chromatography (GC) – the separation of a mixture of compounds intoseparate components, which then can be analyzed by a mass spectrometer. TheGC has two main components: stationary phase (solid) and mobile phase (gaseoussamples). The gaseous samples are separated based on their different adsorptionability to the solid phase.12. In some cases, the subject of MS interest is the presence of specific substancesand/or measure of their quantities, e.g. quantitation of low concentratedadmixtures in complex mixtures. In that case the mass spectrometer is set tomonitor only m/z values of ions representative of the molecules of interest.13. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry(MALDI-TOF MS) is a relatively novel technique in which a co-precipitate of anUV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laserpulse. Most of the laser energy is absorbed by the matrix, which preventsunwanted fragmentation of the biomolecule. The ionized biomolecules areaccelerated in an