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An introduction to Mass spectrometry. What is mass spectrometry? Analytical tool measuring molecular weight (MW) of sample Only picomolar concentrations.

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Presentation on theme: "An introduction to Mass spectrometry. What is mass spectrometry? Analytical tool measuring molecular weight (MW) of sample Only picomolar concentrations."— Presentation transcript:

1 An introduction to Mass spectrometry

2 What is mass spectrometry? Analytical tool measuring molecular weight (MW) of sample Only picomolar concentrations required Within an accuracy of 0.01% of total weight of sample

3 History of Mass spectrometry 1953 : Quadrupole and the ion trap(W. Paul at H.S. Steinwedel). Nobel Prize to Paul in 1989. 1956 : First GC-MS 1968 : First commercial quadrupole 1975 : First commercial GC-MS 1990s : Explosive growth in biological MS, due to ESI & MALDI 2002 : Nobel Prize to Fenn & Tanaka for ESI & MALDI 2005 : Commercialization of Orbitrap MS

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5 Inlet Ion Source Mass Analyzer Detector Data System High Vacuum System MALDI ESI FAB LSIMS EI CI HPLC Flow injection Sample plate Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS Microchannel plate Photomultiplier Electron multiplier Simplified Schematic

6 Principle : If ions are accelerated with the same potential at a fixed point and a fixed initial time and are allowed to drift, the ions will separate according to their mass-to-charge ratios.

7 The ions enter the flight tube with the lighter ions traveling faster than the heavier ions The lighter ions strike the detector before the heavier ions. This “time of flight”(TOF) can be converted to mass

8 Where are they used? Biotechnology : analysis of proteins, peptides, oligonucleotides Pharmaceutical : drugs discovery, combinatorial chemistry, pharmokinetics, drug metabolism Clinical : neonatal screening, haemoglobin analysis, drug testing Environmental : water, food, air quality (PCBs etc) Geological : oil composition

9 In biochemistry Accurate molecular weight measurements : purity of sample, detection of amino acid substitutions, post-translational modifications, and disulphide bridges Reaction monitoring : enzyme activity, chemical modification, protein digestion Amino acid sequencing Oligonucleotide sequencing Protein structure : protein folding (H/D exchange), protein-ligand complex formation, macromolecular structure determination

10 How does a Mass Spectrometer work? Three fundamental parts: the ionisation source, the analyser, the detector Samples easier to manipulate if ionised Separation in analyser according to mass-to-charge ratios (m/z) Detection of separated ions and their relative abundance Signals sent to data system and formatted in a m/z spectrum

11 The mass spectrum shows the results Relative Abundance Mass (m/z) 0 10000 20000 30000 40000 50000 100000 150000 200000 MH + (M+2H) 2+ (M+3H) 3+ MALDI TOF spectrum of IgG

12 H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ [M + 5H] 5+ [M + 4H] 4+ [M + 3H] 3+ [M + 2H] 2+ [M + 1H] + M/Z = 10,003 / 3 M/Z = 3334 M/Z = 10,005 / 5 M/Z = 2001 M/Z = 10,004 / 4 M/Z = 2501 M/Z = 10,002 / 2 M/Z = 5001 M/Z = 10,001 / 1 M/Z = 10,001 Relative Intensity(%) 02500500010000 5+ 2001 4+ 2501 3+ 3334 2+ 5001 1+ 10001 The same protein with a molecular weight of 10,000 contains 5, 4, 3, 2, and 1 charges

13 1981.84 1982.84 1983.84 Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) No 13 C atoms (all 12 C) One 13 C atom Two 13 C atoms “Monoisotopic mass”

14 Important performance factors 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 detected / analyzed?

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16 Mass measurement accuracy depends on resolution 0 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Resolution = 14200 Resolution = 4500 Resolution =18100 15 ppm error 24 ppm error 55 ppm error High resolution means better mass accuracy

17 MALDI TOF training Procedure [introduction] Mass Spectrometry Peptide Mass Fingerprinting [Summary] MALDI-TOF MS System MALDI-TOF MS sample MALDI-TOF MS Data acquisition.

18 Mass spectrometry? Ionizer Gas Phase Sample + _ Mass Analyzer Detector MALDI Electro-Spray Ionization (ESI) Time-Of-Flight (TOF) Quadrapole Ion-Trap Electron Multiplier (EM)

19 Matrix Assisted Laser Desorption Ionization (MALDI)

20 Time-of-Flight (TOF) amp comp L+L+ M+M+ H+H+ +20 kV +22 kV 0 kV 1) Ions enter source region, accelerated toward reflectron. 2) Ions separate in space based on their relative mass-to-charge (m/z). 3) Ions reverse path in reflectron. 4) Ions impact detector. Flight time Signal [Molecular weight]

21 Mass spectrometry

22 Peptide Mass Fingerprinting Trypsin NK K K K K K R R R R C N C K K K K K K R R R R Protein Tryptic peptide mixture. Masses measured by MS. Every peptide has a basic C-terminus. A protein can be identified in a database by matching masses of a subset of the tryptic peptides against calculated values.

23 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVS PFDHSRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVW EQKSRGVVMLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIK SYYTVRQLELENLTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRE SGSLSPEHGPVVVHCSAGIGRSGTFCLADTCLLLMDKRKDPSSVDIKKVL LEMRKFRMGLIQTADQLRFSYLAVIEGAKFIMGDSSVQDQWKELSHEDLE PPPEHIPPPPRPPKRILEPHNGKCREFFPNHQWVKEETQEDKDCPIKEEK GSPLNAAPYGIESMSQDTEVRSRVVGGSLRGAQAASPAKGEPSLPEKDED HALSYWKPFLVNMCVATVLTAGAYLCYRFLFNSNT intact protein enzyme peptide fragments

24 Peptide Mass Fingerprinting 2D-Gel In Gel Digestion MS 848.1 1272.5 492.6 883.2 2978.9 812.6 1432.3 3127.1 996.8 702.4 164.9 2748.2 848.3 1272.7 493.2 882.6 2978.3 364.1 948.9 3128.8 Database 3514.2 2837.1 263.9 147.4 1429.7 199.6 142.3 640.8 Is identical to In Silico Digestion

25 1.MALDI-TOF MS System MALDI-TOF MS (Applied Biosystems, Voyager STR-DE)

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27 - MALDI-TOF MS Experiment - 1.MALDI-TOF MS System

28 2. MALDI-TOF MS Sample Plate : 100 or 96 X 2 well Matrix : Alpha-cyano-4-hydroxycinnamic acid, THAP, Sinapinic acid Sample : Large protein Peptide [Protein Mass Fingerprinting] Small molecules Lipid, Carbohydrate, Oligonucleotides

29 2. MALDI-TOF MS Sample -MALDI-TOF MS matrix-

30 2. MALDI-TOF MS Sample -Matrix Crystal-

31  Peptide Sample preparation Excise Wash Extract Dry gel Reconstruction MS Digest 2. MALDI-TOF MS Sample -Protein identification-

32 3. MALDI-TOF MS Data acquisition a. Instrument control panel b. Data explorer

33 3. MALDI-TOF MS Data acquisition

34 SEE INSTRUMENT CONTROL PANEL!!! 3. MALDI-TOF MS Data acquisition

35 Angiotensin 1 - 1295.6675(monoisotopic mass) Renine - 1758.9331 (monoisotopic mass) ACTH - 2465.1989 (monoisotopic mass) ★ About 10 pmol 3. MALDI-TOF MS Data acquisition -MALDI-TOF MS calibration-

36 Run instrument control panel Insert plate Load plate *.bic file load High voltage on calibration Start acquisition Save spectrum Post data acquisition 3. MALDI-TOF MS Data acquisition -MALDI-TOF MS Experiment-

37 Procedure of *.PKT file 1.Data acquisition (*.dat file) 2.Baseline correction 3.Noise remove 4.De-isotope 5.Save as peak [mouse right-button click on result window] 3. MALDI-TOF MS Data acquisition - Post Data acquisition-

38 3. MALDI-TOF MS Data acquisition -Data Search [profound; Mascot; MS-Fit] -

39 Desalting method 1) 샘플을 C18 ZipTip 에 4 ∼ 5 회 pipetting 2)20 ㎕ 0.1%TFA 로 2 ∼ 3 회 pipetting 3)3 ㎕ Matrix solution(in 50% ACN) 으로 Elution( 이때 직접 MALDI plate 에 Loading) Standard loading 1)Stadard+Matrix tube 에서 0.5 ㎕씩 Loading.


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