Mass Spectrometry for Chemists and Biochemists
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1 Erasmus Intensive Program SYNAPS Univ. of Crete - Summer 2007 Mass Spectrometry for Chemists and Biochemists Spiros A. Pergantis Assistant Professor of Analytical Chemistry Department of Chemistry University of Crete spergantis@chemistry.uoc.gr Tel Office Number: A206 1 Module Outline What is Mass Spectrometry (MS) and what is it used for? Ionization Sources Analyzers Detectors Tue Modes of fragmentation in MS Applications: Identification of organic compounds, proteomics, environmental analysis. Hyphenated techniques (LC-MS and GC-MS) Quantitation with MS Problem Set 2 1
2 Ionization Ion Source Form ions (charged molecules) Mass Sorting (filtering) Mass Analyzer Sort Ions by Mass (m/z) Detection Ion Detector Detect ions Inlet Solid Liquid Vapor Mass Spectrum 3 Concepts of mass Assigning numerical value to the intrinsic property of mass is based on using carbon-12, 12 C, as a reference point. One unit of mass is defined as a Dalton (Da). One Dalton is defined as 1/12 the mass of a single carbon-12 atom. Thus, one 12 C atom has a mass of Da. 4 2
3 Isotopes + Most elements have more than one stable isotope. For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da. + Why do we care? Mass spectrometers can see isotope peaks if their resolution is high enough. If a MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved. 5 Stable isotopes of most abundant elements of organic compounds Element Nominal mass H 1 2 C N O Exact Mass Rel. Ab. (%) % Average Rel. Ab. Mass (%) % Mass Decane Leuenkephalin Bovine proinsulin C 10 H 22 C 28 H 38 N 5 O 7 C 381 H 586 N 107 O 114 S 6 Nominal Exact Average
4 Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) Monoisotopic mass No 13 C atoms (all 12 C) One 13 C atom Two 13 C atoms 7 Isotope pattern for a larger peptide (207 C-atoms) m/z 8 4
5 Mass spectrum of insulin 2 x 13 C 13 C 12 C : Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used. 9 Monoisotopic mass Monoisotopic mass corresponds to lowest mass peak When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement. 10 5
6 Average mass Average mass corresponds to the centroid of the unresolved peak cluster When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass. 11 What if the resolution is not so good? At lower resolution, the mass measured is the average mass. Better resolution Poorer resolution Mass 12 6
7 ISO:CH3 How is mass resolution calculated? M R = M/ M % Intensity FWHM = M Mass (m/z) Example Calculate the resolution required to separate CO and N
8 Mass measurement accuracy depends on resolution High resolution means better mass accuracy ppm error Resolution =18100 Counts ppm error Resolution = ppm error Resolution = Mass (m/z) 15 Important performance characteristics of mass analyzers Mass accuracy: How accurate is the mass measurement? Resolution: How well separated are the peaks from each other? Sensitivity: How small an amount can be analyzed? 16 8
9 Mass Analyzers Quadrupole Mass Filter Linear Time-of-Flight (TOF) ReflectronTOF Double Focusing Magnetic Sector Quadrupole Ion Trap Fourier Transform Ion Cyclotron Resonance (FT-ICR-MS) 17 Mass analyzers separate ions based on their mass-to-charge ratio (m/z) Operate under high vacuum (keeps ions from bumping into gas molecules) Actually measure mass-to-charge ratio of ions (m/z) Key specifications are resolution, mass measurement accuracy, and sensitivity. 18 9
10 Quadrupole mass analyzer cm Has four parallel metal rods. 1 cm 19 Quadrupole cross section Uses a combination of and DC voltages to operate as a mass filter. +DC (180 o out of phas 20 -DC 10
11 Quadruple mass analyzer L = 250 mm +DC Lets one mass pass through at a time. -DC 21 Quadruple mass analyzer L = 250 mm +DC -DC 22 11
12 Quadruple mass analyzer L = 250 mm Can scan through all masses or sit at one fixed mass. +DC -DC 23 Quadrupoles have variable ion transmission modes m2 m4 m1 m3 m4 m3 m2 m1 mass scanning mode m2 m4 m1 m3 m2 m2 m2 m2 single mass transmission mode 24 12
13 x z y 25 Difficult to defocus a heavy ion x + DC z DC 1,5 1 0, ,5-1 -1,5 Χρόνος 26 13
14 x + DC z DC Intensity m/z 27 Difficult to refocus a heavy ion z -DC + -DC 1,5 y 1 0, ,5-1 -1,5 Χρόνος 28 14
15 Difficult to refocus a heavy ion z -DC + -DC y Intensity m/z 29 Stability diagram a φ Unstable oscillation m 1 DC m 2 Stable oscillation m 2 q φ m
16 Ion stability during scanning a φ /DC = const DC low m high m q φ 31 Quadrupole Mass Filter Advantages Inexpensive Easily Interfaced to Many Ionization Methods Disadvantages Low Resolution (<4000) Low Accuracy (>100ppm) MS/MS requires multiple analyzers Low Mass Range (<4000) Slow Scanning 32 16
17 Time-of-Flight (TOF) mass analyzer Source Drift tube detector D Ions are introduced in pulses. The drift region is field free. Measures the time for ions to reach the detector. Small ions reach the detector before large ones. 33 Linear Time-of-Flight (TOF) Advantages Extremely High Mass Range (>1 MDa) Fast spectral acquisition Disadvantages Low Resolution (4000) Low Accuracy (>200ppm) MS/MS not possible 34 17
18 Reflectron Time-of-Flight (TOF) Advantages High Resolution (>20,000 in some models) High Accuracy (<5ppm) 10,000 Mass Range Fast Spectral aquisition Disadvantages 35 Reflectron Time-of-Flight (TOF) Courtesy Bruker Datonics BioTOF user s Manual 36 18
19 Double-Focusing Magnetic Sector Advantages Very High Resolution (60,000) High Accuracy (<5 ppm) 10,000 Mass Range Disadvantages Expensive Requires Skilled Operator 37 Ion Trap Mass Analyzer Cut away side view 38 19
20 Ion trap geometry Ring Electrode Entrance cap End cap 39 Primary Auxiliary Skimmer
21 Quadrupole Ion Trap Advantages Inexpensive Easily Interfaced to Many Ionization Methods MS/MS in one analyzer Disadvantages Low Resolution (<4000) Low Accuracy (>100ppm) Space Charging Causes Mass Shifts Low Mass Range (<4000) Slow Scanning 41 FT-ICR-MS Advantages Extremely High Resolution (>500,000) Very Good Accuracy (<1 ppm) MS/MS in one analyzer Disadvantages Expensive Requires Superconducting Magnet Slow MS/MS 42 21
22 How do mass spectrometers get their names? Types of ion sources: Electrospray (ESI) Matrix Assisted Laser Desorption Ionization (MALDI) Types of mass analyzers: Quadrupole (Quad, Q) Ion Trap Time-of-Flight (TOF) - MALDI-TOF, ESI-Quad. - ESI-QQQ, ESI Q TOF, ESI Q Trap, MALDI TOF-TOF 43 Ions are detected with a microchannel plate primary ion -1000V + e - L e - e - e- D -100V L >> D 44 22
23 45 23
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