Mass spectrometry basic principles
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(MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the atoms or molecules comprising a sample of material. The spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds,so it is considered one f the very important diagnostic analytical techniques .
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Mass spectrometry basic principles
- 1. MEDICAL RESEARCH INSTITUTE– ALEXANDRIA UNIVERSITY 15 th conference (3 rd international) ADVANCES IN MEDICAL RESEARCH ` From molecular medicine-to clinical application` Mass spectrometric techniques RANIA MOHAMED EL-SHARKAWY Lecturer of clinical chemistry , Medical Research Institute Alexandria University
- 3. Mass spectrometry (MS) Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known materials, and to elucidate the structure and chemical properties of molecules A mass spectrometer is a device that measures the mass-to-charge (m/z) ratio of ions.
- 7. Simple Definition: Ionization is a process of charging a molecule. Molecules must be charged in order to measure them using a mass spectrometer
- 8. Types Of ionizing radiation Important to determine the target from analyte investigated
- 9. Hard ionization: the resulting unique fragmentation patterns can be used as a fingerprint for the identification of the sample. Fragment ions also provide important information about the primary structure . (i.e. sequence) of the sample molecules Soft ionization is applied to a stable molecule, the exact m/z value of its stable molecular thus, allows for the compositional analysis of the sample of unknown structure under .study
- 11. Chemical ionization A proton is transferred to or abstracted from ,a gas phase analyte by a reagent gas molecule Little or no fragmentation as the protonated molecule is not highly excited in the chemical ionization Uses: analyte molecular mass determination & quantification
- 14. The 2002 Nobel Prize in Chemistry was awarded to two mass spectrometrists (J. Fenn and K. Tanaka) for their development ionization of techniques, which include electrospray ionization
- 15. 3-Matrix Assisted Laser Desorption Ionization (MALDI)
- 16. SELDI Modification of MALDI surface a type of affinitycapture property Sample of interest is exposed to affinity surface ,certain analyte will preferentially bind Washing to remove excessMatrix is added to asset ionizationUses: analysis of protein mixture with low sample size (purification&analysis occur on the same surface
- 18. II. Analysis and Separation of Sample Ions
- 19. The mass analyzer separates the ions formed in the ionization source according to their mass-to-charge (m/z) ratios using some physical property e.g. electric or magnetic fields.
- 20. Time Of Flight Mass Analyzer (TOF)
- 22. . III. Detection of sample ions
- 23. • A tiny current is produced when the ion reaches the detector. • The detector amplifies the signals which are then transmitted to the data system to be represented as peaks on a mass spectrum Mass spectrum: graphic representation of ions separated according to their m/z ratio
- 25. Mass spectrum of CO2
- 26. Mass spectrum Questions needs to be asked?? 1.Negative or positive mode 2.Types of ion source result in different arrays of fragments produced from the original molecules. 3.origin of a sample 4. How the sample was prepared ??
- 27. Interpretation of mass spectra • compare its experimental mass spectrum against a library of mass spectra library of mass spectra • If the search comes up empty, then manual interpretation or software assisted interpretation of mass spectra are performed •A recent technique for structure elucidation in mass spectrometry, called precursor ion fingerprinting identifies individual pieces of structural information by conducting a search of the tandem spectra of the molecule under investigation against a library of the product-ion spectra of structurally characterized precursor
- 29. Techniques Of mass spectrometry • GC –MS : 1.Definitive method to quantify standard reference materials 2.Identifying trace contaminants or toxins 3.Identification of drugs
- 30. Techniques Of mass spectrometry • HPLC –MS : 1.Screening & confirmation in inborn errors of metabolism 2.Analysis of amino acids
- 31. MALDI
- 32. SELDI-TOF
- 33. Tandem Mass Spectrometry A tandem mass spectrometer is a mass spectrometer that has more than one analyser, usually two, with a collision chamber in between.
- 34. MS/MS uses 2 stages of mass analysis: 1. Selection of an ion 2. Analysis of ion fragments produced by collision with inert gas.
- 35. Phe Subs B
- 36. APPLICATIONS OF mass spectrometry Pharmacokinetics New born screening Proteomics and mass spectrometry
- 37. applications of mass spectrometry to study proteins Best two techniques: •ESI •MALDI Using two approaches: •Top-Down •Bottom-Up
- 39. MS/MS allows the screening of a numerous array of metabolic disorders in a single analytical run: • Amino Acid Disorders PKU, MSUD, Tyr, Cit, etc • Fatty Acid Oxidation Disorders MCAD, VLCAD, LCHAD, etc. • Organic Acid Disorders PA, MMA, IVA, GA-1, etc
- 40. intensity%
- 41. Advantages of MS/MS as a screening tool in NBS Sensitive Specific Accurate Quantitation Internal standards: gold standard for accuracy High impact Multiple Metabolite, Multiple Disease Screening cost effective High throughput
- 42. applications of mass spectrometry to study proteins Best techniques: •ESI - Quadrupole •MALDI-TOF •SELDI-TOF
- 43. TOP-DOWN approach intact proteins are ionized by either of the three techniques described above, and then introduced to a mass analyzer
- 44. BOTTOM-UP approach Bottom-up proteomics is a common method to identify proteins and characterize their amino acid sequences and post-translational modifications by proteolytic digestion of proteins prior to analysis by mass spectrometry.
- 45. applications of mass spectrometry to study proteins Identifying unknown proteins Protein sequencing ( peptides are sequenced by generating multiple sets of peptides)
- 46. applications of mass spectrometry to study proteins Identification of chemical modification ( post-translational modification in proteins after synthesis) Identification of organisms (identifying bacteria by finger printing proteins)
Editor's Notes
- First The sample has to be introduced into the ionisation source of the instrument. Once inside the ionisation source, the sample molecules are ionised, because ions are easier to manipulate than neutral molecules. The formedn ions enter the mass analyzer where they are sorted according to their mass (m) -to-charge (z) ratios (m/z). the ions encountered in Mass Spectrometry have just one charge (z=1) so the m/z value is numerically equal to the molecular (ionic) mass in Daltons. The sorted ions reach the detector where they are detected and sent to the data system for analysis and representation in the format of a m/z spectrum. The analyser and detector of the mass spectrometer, and often the ionisation source too, are maintained under high vacuum to give the ions a reasonable chance of travelling from one end of the instrument to the other without any hindrance from air molecules
- Charged particles easier to manipulate than neutral
- MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation. The sample is pre-mixed with a highly absorbing matrix compound. The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture.
- A mass analyzer uses some physical property (e.g., electric or magnetic fields or time of flight) to separate ions of a particular m/z value, The main function of the mass analyser is to separate , or resolve , the ions formed in the ionisation source of the mass spectrometer according to their mass-to-charge (m/z) ratios.
- The time-of-flight analyser separates ions by measuring the time it takes ions to travel through an air-free region known as the flight tube. Poor mass resolution Heavier ions are slower than lighter ones.
- Quadrupole mass analyzers consist of four rods with a circular or hyperbolic cross section. Each pair of opposing rods is either positively or negatively charged (Fig. 2 ). The ions entering the analyzer are separated according to their mass-to-charge ratio based on their trajectories when exposed to the electric field in the space between the rods. oscillate in an electric field (the quadrupole field) between the paired rods of the quadrupole. By changing the characteristics of the field, ions can be manipulated & molecules with a specific m/z ratio (molecule A+) will oscillate with a harmonic ion trajectory creating an ion beam that traverses the quadrupole. All other ions (molecule B+) are filtered out of the ion beam.
- The m/z values of the ions are plotted against their intensities to show the number of components in the sample, the molecular mass of each component, and the relative abundance of the various components in the sample.
- MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation. The sample is pre-mixed with a highly absorbing matrix compound. The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture.
- After being prepped, the sample is injected into the first instrument. While in the first instrument, the sample is ionized to produce molecular ions and the type of molecules present are determined based upon mass-to-charge (m/z) ratio. The entire process, from ionization and sample injection to data acquisition takes only seconds. The ionized molecules are sorted and weighed. Afterward, the sample is sent into the collision cell chamber. In the collision cell chamber, the molecular ion sample is broken into fragmented pieces, called analytes, which are like pieces of a puzzle. After being fragmented, the sample is passed into the second instrument. Within the second instrument, quantities of the selected analyte (s) are sorted and weighed according to their m/z ratio. The peak of each analyte is compared to internal standard to yield both a qualitative and quantitative result .The result can be analyzed within minutes using sophisticated computer programs to produce histograms for analysis.