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Lecture 9 Mass Spectrometry Oct 2011 SDMBT October 2011 SDMBT.

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Presentation on theme: "Lecture 9 Mass Spectrometry Oct 2011 SDMBT October 2011 SDMBT."— Presentation transcript:

1 Lecture 9 Mass Spectrometry Oct 2011 SDMBT October 2011 SDMBT

2 Objectives Describe the principle of Mass Spectrometry
In particular, the ionisation methods (a) MALDI (b) ESI Oct 2011 SDMBT October 2011 SDMBT

3 General workflow for proteomic analysis
Sample Sample preparation Protein mixture Sample separation and visualisation Comparative analysis Digestion Peptides Mass spectrometry MS data Database search Protein identification Oct 2011 SDMBT October 2011 SDMBT

4 Protein Identification
MALDI-TOF ESI-MS Molecular weight of tryptic peptides Molecular weight of protein MS/MS Molecular weight of peptide fragments Oct 2011 SDMBT

5 Mass-to-charge ratio (m/z)
Mass spectrometry workflow Typical process ionisation detection Mass-to-charge ratio (m/z) acceleration sorting (Medical College of Georgia) Oct 2011 SDMBT October 2011 SDMBT

6 The need for ionisation
Analyte has to be converted into gas-phase ions – only charged ions can be detected by MS Movement of gas-phase ions can be precisely controlled by electromagnetic fields Difficulty of generating gas-phase ions results in complex instruments and high costs – need high vacuum Gas phase ions can be generated by e.g. an electron beam (electron ionisation) collision with inert gas molecules (MS-MS) a laser beam (MALDI-TOF) a charged nozzle (ESI-MS) Oct 2011 SDMBT October 2011 SDMBT

7 Mass Spectrum A plot mass of ions (m/z) (x-axis) versus the intensity of the signal (roughly corresponding to the number of ions) (y-axis) Mass spectrum of water – ionised by electron ionisation Notice that the water molecule fragments upon ionisation H2O+ HO+ O+ H+ Oct 2011 SDMBT

8 Notice: the horizontal axis is mass/charge ratio of ions (m/z)
HCHO+ CHO+ CO+ HCHO2+ HCH+ CH+ C+ Notice: the horizontal axis is mass/charge ratio of ions (m/z) Oct 2011 SDMBT

9 The need for ionisation
Proteins are large macromolecules – electron ionisation works only for volatile compounds – also electron ionisation is a ‘hard’ ionisation technique – molecule fragments into smaller ions Need to find an efficient method to convert proteins from liquid phase to gas-phase ions Soft ionisation methods like electrospray ionisation (ESI) and matrix-assisted laser-desorption ionisation (MALDI) Oct 2011 SDMBT October 2011 SDMBT

10 Soft ionisation Ionise peptides into gas-phase ions
Too much energy (hard ionisation) breaks peptides into smaller fragments (Prof. Jose-Luis Jimenez) Oct 2011 SDMBT October 2011 SDMBT

11 Soft ionisation MALDI-TOF mass spectrum Peptide does not fragment
Maldi TOF/TOF mass spectroscopic spectre for UCH-L3 digested Ecotin-Ubiquitin-FLS in the m/z range 799–4013. The peaks at 2683 and 1342 represent the single and double protonation states of the FLS peptide, respectively. Oct 2011 SDMBT

12 MALDI Peptides are mixed with a matrix, which crystallise on a metal plate Peptides protonated by matrix and solvent Pulses of laser transfer energy to matrix Matrix vaporises with peptide samples Oct 2011 SDMBT

13 MALDI Oct 2011 SDMBT

14 MALDI plate (University of Pittsburgh BRSF) (Bruker Daltonics)
Oct 2011 SDMBT October 2011 SDMBT

15 Matrix Contains ring structures to absorb energy from UV laser
Contains acid group to protonate peptides Matrix solution – DHB or CHCA dissolved in acetonitrile and trifluoroacetic acid Sinapinic acid a-cyano-4-hydroxycinnamic acid Oct 2011 SDMBT October 2011 SDMBT

16 Characteristics of MALDI
Efficient protonation of peptides Ions generated in discrete packets due to laser pulses Combined with time-of-flight (TOF) mass analysis to give very high sensitivity More tolerant than ESI to contaminants (urea, SDS), thus HPLC separation is not required Oct 2011 SDMBT

17 MALDI-TOF of mixture of cytochrome, ubiquitin, myoglobin
Mw of cytochrome C 12000                                                                             Mw of ubiqutin 8500 Mw of myoglobin 17000 MALDI-TOF of mixture of cytochrome, ubiquitin, myoglobin (without PSD) – note single peaks (except cytochrome) – can tell Mw of 3 proteins in one spectrum Oct 2011 SDMBT

18 Trypsin digested peptides
From last lecture: one spot in 2D-gel represents one protein – this protein is digested with trypsin into smaller peptides. The peptide mixture is spotted on the MALDI plate Arginine or Lysine Proline (ExPasy PeptideCutter) Oct 2011 SDMBT

19 MALDI spectrum of Tryptic digest of β-casein
Major peaks at: Each peak represents one peptide digested by trypsin from casein Represent expected sizes of tryptic peptides Soft ionisation ensures no further fragmentation of peptides King’s College London (Pierce) Oct 2011 SDMBT October 2011 SDMBT

20 Tryptic digest of β-casein
Most abundant peak  base peak, abundance set to 100% MS is seldom quantitative, only tells relative abundance Ideally every peptide is ionised to same extent  100% King’s College London (Pierce) Oct 2011 SDMBT

21 Electrospray Ionisation (ESI)
Peptides in acidic solution are pushed out of a fine capillary A high positive charge at the capillary results in the formation of a Taylor cone Peptides protonated in charged droplets and ionised Gas-phase ions repelled from capillary into instrument Nitrogen aids in evaporation of solvent from charged droplets Oct 2011 SDMBT

22 Electrospray Ionisation (ESI)
(Royal Society of Chemistry) Oct 2011 SDMBT

23 Electrospray Ionisation (ESI)
High voltage results in accumulation of positive charges on droplets Increased charge density results in instability of droplets Droplets break down successively into smaller sizes Oct 2011 SDMBT (New Objective, Inc)

24 Characteristics of ESI
Acidic conditions result in protonation of all basic sites in peptides Basic side groups of lysine, arginine, histidine Produces peptide ions that are multiply protonated Advantageous for peptides digested by trypsin as doubly charged peptides are formed Oct 2011 SDMBT

25 Characteristics of ESI
Efficient ionisation process results in sensitivity of ESI experiments General compatibility of ESI with reversed-phase high performance liquid chromatography (RP-HPLC)  LC/MS-MS Oct 2011 SDMBT

26 ESI-MS - often see doubly, triply, multiply charged ions
Oct 2011 SDMBT

27 Different scale on horizontal axis Mw=1413.8x12-12 = 16953.6
ESI of myoglobin Compare with slide 17 More complicated Different scale on horizontal axis Mw=1413.8x12-12 = Mw – need to know how to interpret by assigning charges Oct 2011 SDMBT

28 Different scale on horizontal axis ESI of cytochrome C
Compare with slide 17 More complicated Different scale on horizontal axis ESI of cytochrome C Oct 2011 SDMBT

29 Different scale on horizontal axis
Compare with slide 17 More complicated Different scale on horizontal axis 7+ 6+ 5+ ESI of ubiquitin Mw = Oct 2011 SDMBT

30 MALDI-TOF – 3 peaks Each of each protein ESI – complicated
if sample was a mixture – 3 superimposed spectra Oct 2011 SDMBT

31 Comparison of ionisation methods
MALDI-TOF ESI Sample Solution but ends up embedded in crystalline matrix Sample tolerant to salts Singly charged ions Adduct-formation not so Common. Appearance – one peak Solution eg can come straight from HPLC Results can be affected by salts eg phosphates Multiply-charged ions Adducts with salts common May be difficult to interpret spectra Appearance – many peaks due to multiple charges Oct 2011 SDMBT

32 Comparison of ionisation methods
MALDI-TOF ESI Ionisation Laser Charged spray nozzle Mass analyser TOF Quad Fragmentation is made possible by PSD (Post-Source decay) “Pseudo-MS-MS” MS-MS or Tandem MS Ions fragmented by collision with inert gas Use Mainly peptides Peptides and proteins Oct 2011 SDMBT

33 Components of a mass spectrometer
ionisation acceleration detection Vacuum System Atmosphere Sample Inlet Ionisation Method Mass Analyser Detector Data System MALDI ESI TOF Quadrupole Ion trap Combination Oct 2011 SDMBT (adapted from Thermo Finnigan)

34 The need for a mass analyser
Gas-phase ions has to be filtered/arranged in order to allow selection of specific ions for further analysis. Movement of gas-phase ions precisely controlled by use of electromagnetic fields in a mass analyser Main types of mass analyser TOF (time-of-flight analyser) Quadrupole analyser Fourier-Transform Ion Cyclotron Resonance Orbitrap Oct 2011 SDMBT October 2011 SDMBT

35 TOF Time-of-flight (TOF) analyser is the simplest mass analyser
Peptide ions accelerated into flight-tube, and maintains a velocity due its given kinetic energy TOF analyzer requires that ions are introduced in a pulse  well-suited for MALDI Resolution increases with length of TOF tube Oct 2011 SDMBT

36 Linear TOF (John Lennon, University of Washington) Oct 2011 SDMBT

37 MALDI- TOF with reflectron
Reflectron - This turns ions around in an electric field, sending them towards the detector – improves mass resolution Oct 2011 SDMBT

38 Quadrupole Made up of 4 parallel gold bars
Complex electromagnetic field set up which allows only ions of a a set mass/charge ratio through (NASA) Oct 2011 SDMBT

39 Flight path of an ion through a quadrupole
Correct m/z ratio Larger/smaller m/z ratio Ions that spiral out of control crash into the rods or casing Oct 2011 SDMBT

40 Fourier Transform Ion cyclotron resonance (FT-ICR)
Magnetic fields applied to trapping plates constrain the ions to move around in a circle in between the plates. The circular motion induces an alternating current. The frequency of the AC is related to the m/z ratio. Main advantage of FT-ICR is very high mass resolution See this website Oct 2011 SDMBT

41 Orbitrap Similar to FT-ICR
Electric field applied to trapping plates constrain the ions to move around in a circle in between the plates. The circular motion induces an alternating current. The frequency of the AC is related to the m/z ratio. Outer electrode Main advantage of Orbitrap is very high mass resolution Inner electrode Image from thermo.com Oct 2011 SDMBT

42 Tandem MS (MS/MS) MALDI and ESI are soft ionisation techniques – peptides or proteins do not fragment – useful for molecular weight determination However sometimes fragmentation is useful because it give useful information about the amino acid sequence of peptides (next lecture) Fragmentation can achieved by a number of ways Post source decay (PSD) Collision induced dissociation (CID) Infrared multiphoton dissociation Electron capture dissociation (ECD) Electron transfer dissociation (ETD) Oct 2011 SDMBT

43 Post-Source Decay (PSD)
- Method of fragmenting intact peptides - get amino acid sequence information (see next lecture) Use higher laser power or introduce inert gas to collide with ions so that ions fragments between source and TOF - Electronics to select ions to go into the TOF Oct 2011 SDMBT

44 Ion trap All other dissociation techniques involve the use of an ion trap Ion trap essentially a quadrupole where the magnetic field is set such that a particular ion is trapped in the space between the electrodes (NASA) (K. Yoshinari, Rapid Commun. Mass Spectrom. 14, , 2000) Oct 2011 SDMBT

45 Ion trap Collision induced dissociation (CID) – inert gas (e.g. Xe, Ar, N2 or He) is introduced into trap, collisions will cause peptides to fragment usually the C-N peptide bond to produce b and y ions (see later) Infrared multiphoton dissociation (IRMPD) – IR laser is fired into ions to excite the vibrations of peptides. Electron capture dissociation (ECD) – electron beam is fired into ions to produce c and z ions Electron transfer dissociation (ETD) – singly charged anion, fluoranthracene is introduced. An electron is transferred to peptide. Results in c and z fragments Oct 2011 SDMBT (March, JMS Vol. 32, , 1997 )

46 Hybrid mass analysers Basically a combination of two types of mass analysers or many of the same type Each analyser performs a different function Used to perform tandem mass spectrometry, using collision induced dissociation (CID) Oct 2011 SDMBT

47 Tandem quadrupoles (QQQ/TSQ)
Scanning of all m/z of daughter ions Select specific parent ion CID with inert gas Video Quantitative Chemical Analysis Oct 2011 SDMBT

48 Tandem mass spectrometry
A) MS spectrum of HSP27. The peptide whose MS/MS spectrum is shown in panel B is indicated. B) MS/MS spectrum of the peptide ion m/z 1163 obtained in CID mode. Perroud et al. Molecular Cancer :64 Oct 2011 SDMBT


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