Presentation is loading. Please wait.

Presentation is loading. Please wait.

Oct 2011 SDMBT Lecture 9 Mass Spectrometry. Oct 2011 SDMBT Objectives  Describe the principle of Mass Spectrometry  In particular, the ionisation methods.

Similar presentations


Presentation on theme: "Oct 2011 SDMBT Lecture 9 Mass Spectrometry. Oct 2011 SDMBT Objectives  Describe the principle of Mass Spectrometry  In particular, the ionisation methods."— Presentation transcript:

1 Oct 2011 SDMBT Lecture 9 Mass Spectrometry

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

3 Oct 2011 SDMBT 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

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

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

6 Oct 2011 SDMBT 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)

7 Oct 2011 SDMBT 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 Mass Spectrum H 2 O + HO + O + H +

8 Oct 2011 SDMBT HCHO + CHO + CO + HCHO 2+ HCH + CH + C + Notice: the horizontal axis is mass/charge ratio of ions (m/z)

9 Oct 2011 SDMBT 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)

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

11 Oct 2011 SDMBT 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. MALDI-TOF mass spectrum Peptide does not fragment Soft ionisation

12 Oct 2011 SDMBT 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

13 Oct 2011 SDMBT MALDI

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

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

16 Oct 2011 SDMBT 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

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

18 Trypsin digested peptides (ExPasy PeptideCutter) Arginine or LysineProline 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

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

20 Oct 2011 SDMBT Tryptic digest of β-casein King’s College London (Pierce)  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%

21 Oct 2011 SDMBT 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

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

23 Oct 2011 SDMBT (New Objective, Inc)  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 Electrospray Ionisation (ESI)

24 Oct 2011 SDMBT 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

25 Oct 2011 SDMBT 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

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

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

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

29 ESI of ubiquitin 5+ Mw = Compare with slide 17 More complicated Different scale on horizontal axis 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 MALDI-TOFESI SampleSolution 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 Comparison of ionisation methods

32 Oct 2011 SDMBT MALDI-TOFESI IonisationLaserCharged spray nozzle Mass analyserTOFQuad Fragmentation is made possible by PSD (Post-Source decay) “Pseudo-MS-MS” MS-MS or Tandem MS Ions fragmented by collision with inert gas UseMainly peptidesPeptides and proteins Comparison of ionisation methods

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

34 Oct 2011 SDMBT 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

35 Oct 2011 SDMBT 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

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

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

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

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

40 Oct 2011 SDMBT Fourier Transform Ion cyclotron resonance (FT-ICR) See this website 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

41 Oct 2011 SDMBT Orbitrap Image from thermo.com 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. Inner electrode Outer electrode Main advantage of Orbitrap is very high mass resolution

42 Oct 2011 SDMBT 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)

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

44 Oct 2011 SDMBT (K. Yoshinari, Rapid Commun. Mass Spectrom. 14, , 2000) Ion trap (NASA) 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

45 Oct 2011 SDMBT Ion trap (March, JMS Vol. 32, , 1997 ) -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

46 Oct 2011 SDMBT 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)

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

48 Oct 2011 SDMBT 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


Download ppt "Oct 2011 SDMBT Lecture 9 Mass Spectrometry. Oct 2011 SDMBT Objectives  Describe the principle of Mass Spectrometry  In particular, the ionisation methods."

Similar presentations


Ads by Google