MALDI-TOF mass spectrometry: Theory and principles

1. Rio, Ferbruary 6, 2006 MALDI-TOF mass spectrometry:Theory and principles Peter Roepstorff Protein Research Group Department of Biochemistry and Molecular Biology University of Southern Denmark roe@bmb.sdu.dk www.protein.sdu.dk

2. mass analyser (TOF) ion source (MALDI) detector Computer t (m/z) Matrix-assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight (TOF) mass spectrometer

3. matrix molecules analyte molecules MALDI ”solid solution” deposit of analyte doped matrix crystals

4. Important properties of the MALDI matrix • Strong absorption at the laser wavelength • Homogeneous solid-state mixing with the analyte • Ability to undergo photochemical reaction leading to proton transfer to or from analyte

5. The matrix serves two major functions 1. Dilute and isolate analyte molecules. -Prevents their interaction. 2. Absorption of energy from the laser. -Minimizes sample damage -Efficient energy transfer to the analyte

6. MASS ANALYSER MATRIX IONS AND NEUTRALS LASER + - - ANALYTE IONS + ANALYTE MOLECULES MATRIX MOLECULES Explosive phase transition. Gas jet of matrix and analyte molecules

7. mass analyser (TOF) ion source (MALDI) detector Computer t (m/z) Matrix-assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight (TOF) mass spectrometer

8. 20 kV flight tube (10-8 – 10-9 torr) detector ion source mass: > > t (m/z) Linear time-of-flight mass spectrometer.

9. 20 kV 0 kV + + mv2 2 t0: Ek=0 ta: Ek= q U = • All ions obtain the same kinetic energy • Their final velocity is proportional to(m/q) Ion source Continuous extraction

10. mv2 d d2 Ek= q U = ze U = t = 2Ue v 2 t2 = m/z mass: > > Linear time-of-flight mass spectrometer. 20 kV d

11. 20 kV detector flight tube ion source same mass, but higher initial kinetic energy than t (m/z) -poor resolution ! -Poor mass accuracy  0.5-1 Da Linear time-of-flight mass spectrometer.

12. m resolution= m m=FWHM h m ½ h m

13. Delayed extraction Reflectron Resolution is limited by: Mass resolution is affected by factors creating a distribution in flight times among ions with the same m/z ratio. • The initial time distribution. • The ion production time. • The initial spatial distribution. • Size of the volume where the ions are formed. • The initial energy distribution. • Variation of the initial kinetic energy.

14. laser pulse t = t0 TOF-MS 20 U (kV) : 20 0 Delayed ion extraction (DE)

15. t  tif m/z : = ; V0 ( ) > V0 ( ) U (kV) : 20 20 0 Tif = time of ion formation Delayed ion extraction (DE)

16. t = td U (kV) : 20 18 0 DU = 0.5 kV 19.7 19.2 Td = delayed extraction Delayed ion extraction (DE)

17. V ( ) > V ( ) U (kV) : 20 18 0 + 0.5 kV Delayed ion extraction (DE)

18. V ( ) = V ( ) U (kV) : 20 18 0 + 0.2 kV Delayed ion extraction (DE)

19. V ( ) < V ( ) 18 0 U (kV) : 20 Time focus plane (ideal for ion detection) Delayed ion extraction (DE)

20. Delayed vs. continuous extraction.

21. Additional benefits of DE • Lower level of cheminal noise arising from fragmentation in the ion source. • Reduction of matrix background. • Significant reduction of the dependence of ion flight times on laser intensity.

22. Delayed extraction Reflectron Resolution is limited by: Mass resolution is affected by factors creating a distribution in flight times among ions with the same m/z ratio. • The initial time distribution. • The ion production time. • The initial spatial distribution. • Size of the volume where the ions are formed. • The initial energy distribution. • Variation of the initial kinetic energy.

23. 23 kV 20 kV flight tube Ion source reflector detector same m/z, but higher initial kinetic energy than t (m/z) Good resolution ! Good mass accuracy  0.1-0.3 Da Reflector time-of-flight mass spectrometry

24. The reflectron • Consists of a series of grids and ring electrodes creating a retarding field that acts as an ion mirror. • Corrects the energy dispersion of the ions leaving the source with the same m/z ratio. • Increases the mass resolution at the expense of sensitivity and introduces a mass range limitation.

25. Uvar ,t1 URef time focus plane (virtual source) Uacc High resolution! Mass accuracy ± 10-50 ppm Sensitivity (low femto-mole) t (m/z) DE-MALDI-rTOF-MS

26. Continuous extraction • ÷ Delayed extraction • ÷ Reflector Resolution = 500 • Linear mode • + Delayed extraction • ÷Reflector Resolution = 3,500 • Reflector mode • ÷Delayed extraction • + Reflector Resolution = 10,000 m/z = 1570.68 • Reflector mode • + Delayed extraction • + Reflector Resolution = 20,000 Improvement of resolution.MS of (Glu)-fibrinopeptide B

27. mass analyser (TOF) ion source (MALDI) detector Computer t (m/z) Matrix-assisted Laser Desorption/Ionization (MALDI) Time-Of-Flight (TOF) mass spectrometer

28. Dual microchannel plate detector 0 kV 0 -1.6 -0.9 -0.8 -0.1 oscilloscope Array of lead glass tubes e- e- Ion detection. Secondary Electron Multipliers (SEM) Micro-channel plates (MCP) Electrons Amplification Ions Oscilloscope Computer

29. Advantages of MALDI TOF MS • Theoretical unlimited mass range • TOF MS is fast and sensitive • Compatible with pulsed ion sources • The high resolution of modern instruments results in a mass accuracy of 10-100 ppm • Applicable to a broad range of biopolymers and complex mixture. • Structural information can be obtained by MS/MS analysis

30. CID Detector Source Mass analyser 1 Mass analyser 2 • Precursor selection • - TOF • - Quadrupole • Fragment determination • -TOF MS/MS with TOF instruments. • MALDI TOF/TOF • MALDI Q/TOF

31. MALDI Q-TOF from Micromass.

32. Sample Plate Reflector Detector Laser Reflector CID Cell Source 2 Source 1 V V 1 2 TOF 1 TOF2 4700 TOF/TOF from Applied Biosystems

33. MS/MS Voltages

34. 0 m3 m3 m3 m3 m3 m3 m3 m3 m3 m3 m3 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m2 m1 m1 m1 m1 m1 m1 m1 m1 m1 m1 m1 m1 0 Timed ion selector operation TIS Deceleration + from ion source - TOF 1 to collision cell  Switch down time calculated by low mass gate geometry Switch up time calculated by high mass gate geometry

35. TIS R=1000 is achievable at the laser desorption threshold. In practice R= 200-400 because the laser is operated at elevated intensity to induce LID D. Suckau et al, Anal Bioanal Chem. 2003 Aug;376(7):952-65. Epub 2003 Jun 27.

36. MS/MS MS/MS MS 4 Da Resolution at TIS focus plane Precursor selection.

37. Controlled Ion Fragmentationin the 4700 Two independent mechanisms are involved in the dissociation of the precursors -Metastable decay (PSD) -Collision-induced dissociation (CID) • High-Resolution Data • Control of laser intensity • Control of collision energy (1KeV –3KeV) • Control of collision gas pressure • Four different collision gases

38. Tryp lactogl. 2313 MSMS PSD CID (air)

39. Sensitivity of the 4700 Lactogl. 1245 MSMS (low amol): 30.000 spectra. Lactogl. MS (low amol)

40. Resolution in MS - mode

41. Resolution in MS/MS mode

42. Bruker UltraFlex TOF-TOF

43. MS/MS Voltages

44. 0 0 Timed ion selector operation TIS + from ion source TOF CID - TOF 2 LIFT Switch down time calculated by low mass gate geometry Switch up time calculated by high mass gate geometry Few ns

45. b14 b15 b8 b6 b7 b16 b17 b3 b4 b9 b10 b11 b12 b13 b5 G S H Q I S L D N P D pY Q Q D F F P K y13 y12 y11 y10 y8 y7 y6 y5 y4 y3 y2 y9 -80 (MH+) - 98 (MH+) - 80 b4 y10 y11 b17 b5 y10* b9 y8 b15 y11* y2 y4 b8 b3 b14 b6 y8* b11 y12 b16 y7 b7 y13 b12 b13 y6 y3 b10 y9 [H] y5 MH+ MS/MS of the synthetic phosphopeptide

46. TIS CID Cell V V 1 2

47. MS/MS Voltages Bruker ABI

48. MS and MS/MS of tryptic lactogl. TrypLac 1 pmol, MS 1245.59 TrypLac 1 pmol, 1245.5 MSMS

49. Result of the 1245.58 MS/MS spectrum TPEVDDEALEK, m/z 1245.5 Beta-Lactoglobulin, score 95

50. Advantagesof MALDI MS/MS analysis? • Fast and simple sample preparation. • Only a single sample preparation is needed for PMF and subsequent MS/MS analysis. • It is possible to reanalyze interesting samples. • Sensitive • Relative tolerant towards contaminants as salt • Offline LC MALDI experiments.