Learning Objectives for Mass Spectrometry Lecture

Learning Objectives for Mass Spectrometry Lecture. by Dr. Ilya Ioshikhes, Department of Biomedical Informatics, 3017 Graves Hall, Tel. 292-6514, E-mail: [email protected]

Mass 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 or molecules in which an electrical charge is placed on the molecule and the resulting ions are separated by their mass to charge ratio. 2. The technique of mass spectrometry had its beginnings in J.J. Thomson's vacuum tube where in the early part of the 20th century the existence of electrons and "positive rays" was demonstrated. The primary application of mass spectrometry remained in physics for nearly thirty years. It was used to discover a number of isotopes, 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 later developments in diverse fields ranging from geochronology to biochemical research. 3. Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules. Detection of compounds can be accomplished with very minute quantities (as little as 10-12g, 10-15 moles for a compound of mass 1000 Daltons). This means that compounds can be identified at very low concentrations (one part in 1012) in chemically complex mixtures. Mass spectrometry provides valuable information to a wide range of professionals: physicians, astronomers, and biologists, to name a few. 4. A mass spectrometer is an instrument that measures the masses of individual molecules that have been converted into ions, i.e., molecules that have been electrically 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 an electron 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 mass spectrometry. Early mass spectrometers required sample to be a gas, later applicability of mass spectrometry was extended to liquid and solid samples that commonly 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 is converted to a proportional electrical current. The data system records the magnitude of these electrical signals as a function of m/z and converts this information into a mass spectrum. 8. Resolution or Resolving Power is the ability of a mass spectrometer to distinguish between ions of different mass-to-charge ratios such that greater resolution corresponds directly to the increased ability to differentiate ions. For example, a mass 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 following equation: Resolution = M / ΔM, where M is the m/z of an ion and ΔM is the full-width at half-maximum peak. 9. The mass spectrum of a large molecule typically shows many fragment ions in addition to the molecular ion. The most intense ions have been labeled with their mass-to-charge ratio. The fragment ions are used for deduction of the molecular structures. 10. Mass spectrometry (MS) is often used in combination with separation techniques like gas chromatography (GC), liquid chromatography (LC), capillary electrophoresis and supercritical fluid chromatography (SFC). 11. Gas chromatography (GC) – the separation of a mixture of compounds into separate components, which then can be analyzed by a mass spectrometer. The GC has two main components: stationary phase (solid) and mobile phase (gaseous samples). The gaseous samples are separated based on their different adsorption ability to the solid phase. 12. In some cases, the subject of MS interest is the presence of specific substances and/or measure of their quantities, e.g. quantitation of low concentrated admixtures in complex mixtures. In that case the mass spectrometer is set to monitor 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 an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. The ionized biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between 400 and 350,000 Da. It is a very sensitive method, which allows the detection of low (10-15 to 10-18 mole) quantities of sample with an accuracy of 0.1 - 0.01 %. 14. Generic MS-based proteomics experiment consists of several stages e.g.: 1) Isolation of the proteins of interest from cell and tissue environment; 2) Enzymatic degradation of whole proteins to peptides, usually by trypsin; 3) Separation of peptides by several steps of high-pressure liquid chromatography, with subsequent elution into an electrospray ion source; 4) Tandem MS/MS experiment.