1. Maldi-tof

2. Basics on Voyager..... Basics on Maldi-Tof Basics sample preparation Resolution Delayed Extraction Guide wire/Beam steering Reflector Instrument tuning Calibration Theory

3. Components of a Mass Spectrometer INLETION SOURCEMASS FILTERDETECTOR Sample plate HPLC GC Solids probe MALDI API/Electrospray IonSpray EI, CI TOF Quadrupole Ion Trap Magnetic Sector FTMS Hybrid Microchannel Plate Electron Multiplier

4. Applied Biosystems MALDI-TOF instruments Voyager-DE and DE PROVoyager-DE STR

5. Laser flash produces matrix (M) neutrals, positive, negative ions and sample neutrals. M M*, MH +, (M-H) - Sample molecules (A) are ionised by gas phase proton transfer MH + +A AH + +M (M-H) - +A AH - +M Ion Source: MALDI (Matrix Assisted Laser Desorption Ionisation) + + + +

6. + Ions of same mass, different velocities + + Delayed Extraction (DE) 1: Laser fired. Formed ions detach from plate in the absence of an electric field 0 kV 0 nsec 4: Slow ions catch up with faster ones at the detector +20 kV + + + + 3: Field applied. Gradient accelerates slow ions more than fast ones. +20 kV + + + 2: Expansion of the ion cloud in the absence of an electric field 0 kV 150 nsec + + + Detector

7. Ions with different mass, same Kinetic Energy + + + Detector + + + Flight tube Ions with lighter mass will fly faster and will reach the detector first Detector + + + Flying times of the ions are proportional to m/z ratios + + + Mass Filter: TOF (Time Of Flight) m/z = 2 KE s2s2 t2t2

8. The electrical field applied within the reflector produces an ion mirror effect directing the ions towards a second detector Mass Filter: Reflector TOF Improvement in resolution by Increasing the effective flight length of the tube Re-focusing of analogous ions having slight different energy due to initial spread in the ion source

9. Sample Preparation

10. Laboratory Set-Up

11. Voyager Sample Plates Hydrophobic surface SURFACE TENSION

12. 2,4,6-trihydroxy acetophenone (THAP) -cyano-4-hydroxycinnamic acid 2,5-dihydroxybenzoic acid (2,5-DHB) Dithranol trans-3-indoleacrylic acid Sinapinic acid (3,5-Dimethoxy- 4-hydroxy cinnamic acid) 2-(4-hydroxyphenylazo)- benzoic acid (HABA) 3-hydroxypicolinic acid (3-HPA) Peptide (0.1-10 pmol/ l) Protein (0.1-10 pmol/ l) Oligonucleotide (10-100 pmol/ l) Polymer (10 -4 M) MALDI-TOF Matrices

13. Matrix Preparation and Crystals -cyano Sinapinic acid DHB 5 mg/ml in 50% ACN 0,1% TFA 10 g/L in 30-50% ACN with 0.1% TFA Super-DHB 10 mg/ml in water or 50% ACN A = 10 mg/ml DHB in 20% ACN B = 10 mg/ml 5- methoxysalicylic acid in 50% ACN Combine A:B (9:1)

14. Thin Layer (Acetone) First matrix in acetone (dry) Then sample (dry) On-plate washing possible after drying Thin Layer (Nitrocellulose) NC and matrix solution (dry) Then sample (dry) TFA on top, blow off with an air supply; repeat Dried Droplet First sample Immediately after, matrix in solvent Sandwich Method First matrix Then sample And matrix again (air dry)

15. Isotopic Resolution

16. Resolution - 1 What benefit is high resolution Improved identification of peptides Indication of potential modification Greater degree of mass accuracy Resolution is defined as : Mass / (peak width at half peak height)

17. Resolution - 2 R=M/ M, where M is the mass to of peak and M is the peak width at half maximum. S= peak separation. NOT RESOLVED S= M FULLY RESOLVED M S=2 M

18. Consequence of High Resolution The Grey lines indicate the isotopic distribution of the peptide. The Red line indicates the centroid mass data for each. Resolution 1000 = 1297.000 (Average Mass) Resolution 3000 = 1296.680 (Monoisotopic Mass) RESULT = Better Mass Accuracy

19. High Resolution - Too much data? Monoisotopic resolution of Insulin C 12 : 5730.61 C 13 2 x C 13 In compounds with more than 100 carbon atoms the height of the 13 C isotope peak exceeds the height of the 12 C peak

20. Delayed Extraction

21. NO Delayed extraction + + Acceleration 25KV + + + Fraction of second post laser fire Grid 0% (0KV)Ground + + Acceleration 25KV + + + Ground + + Acceleration 25KV + + + Grid 0% (0KV)Ground

22. Delayed extraction + + + + + Ground + + + + + Fraction of second post laser fire Ground + + Acceleration + + + Ground + + Acceleration + + + Ground + + Acceleration 25KV + + + Grid 60% (15KV)Ground

23. Principle of Delayed Extraction When ions are accelerated they exhibit a broad energy spread. When forming ions in a weak electric field then applying a high voltage pulse after a time delay, this energy spread can be minimized. A potential gradient is formed in the ionization region by the voltages applied to the sample plate and the variable voltage grid. Ref: W.C.Wiley and I.H.McLaren Rev.Sci.Instrum 1953,26,1150-1157

24. Linear mode Reflector mode 10600 10800 11000 11200 11400 11600 m/z continuous extraction R=125 delayed extraction R=1,100 m/z 6130 6140 6150 6160 6170 delayed extraction R=11,000 continuous extraction R=650 Delayed Extraction - Why

25. The Reflector

26. Benefits of Using a Reflector Provides higher performance - resolution and mass accuracy ­ Increases separation due to longer flight time ­ Filters out neutral molecules ­ Corrects time dispersion due to initial kinetic energy distribution Capability for PSD experiment

27. Schematic of Voyager DE-PRO and DE-STR Systems Camera Sample plate Pumping Pumping Beam guide Timed ion selector Reflector Lineardetector Extraction grids Reflector detector Laser

28. What is the reflector? The reflector is an electrical mirror with a voltage potential applied across the sides. The ions are sequentially slowed down through the reflector

29. Velocity Focusing in Reflector Mode Sample plate Ground grid Variable-voltage grid slow fast slow Ions must line up at the beginning of the flight tube Refocusing region - some move farther into reflector, than others Defocusing region This initial focus is refocused by the reflector which can be fine tuned for second order velocity focusing. Slow Fast

30. CALIBRATION THEORY

31. Accuracy, Precision & Resolution a. Precision. This is a measure of repeatability, i.e. the degree of agreement between individual measurements of a set of measurements, all of the same quantity. b. Accuracy. This is a measure of reliability, and is the difference between the True Value of a measured quantity and the Most Probable Value which has been derived from a series of measures. The True Value is, of course, never known. c. Resolution. This is the smallest interval measurable by an instrument CALIBRATION THEORY Definitions

32. CALIBRATION THEORY None of the darts are close to the true value (bulls eye) : the measurements are not accurate. Also, since the darts are not very close to each other, the set of measurements is not precise either. The measurements are all close to the true value, so they are accurate. Also, the measurements are all close to each other, so they are precise Since all of the measurements are close together, they are precise, but since they are not close to the true value, they are not accurate

33. Accuracy : in ppm (or Da, or %) 100 ppm = 0,01% 10 ppm = 0,001% 1 ppm = 0,0001% Resolution : in FWHM (Full Width at Half Maximum) - No unit = M/ M CALIBRATION THEORY Definitions

34. 0 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Res = 14200 Res = 4500 21 ppm error 28 ppm error 55 ppm error Res = 18100 CALIBRATION THEORY Definitions

35. Iterative Calibration Approach 1500 2000 2500 3657.9231 904.4711 1296.6801 1570.6783 2093.0846 2465.2024 1000 1500 2000 2500 3000 3500 1) Calibrate on standards 2) Apply calibration to sample 3) Database search 4) Internally calibrate using hits and resubmit

36. Rank Digest # # (%) SwissProt Species MW (Da) Protein Name 11 20060 4/16 (25%) P15992 YEAST 23748.5 HEAT SHOCK PROTEIN 26.20060 P15992 22 34118 3/16 (18%) P08468 YEAST 94523.6 PET111 PROTEIN PRECURSOR.34118 P08468 22 34931 3/16 (18%) P25301 YEAST 52247.7 DNA REPAIR PROTEIN RAD57.34931 P25301 22 37093 3/16 (18%) P10664 YEAST 38961.1 60S RIBOSOMAL PROTEIN L2A (RP37093 P10664 22 37100 3/16 (18%) P49626 YEAST 38931.0 60S RIBOSOMAL PROTEIN L2B (RP2).37100 P49626 22 54802 3/16 (18%) P38863 YEAST 96825.4 HYPOTHETICAL 96.8 KD PROTEIN54802 P38863 Detailed Results 1. 4/16 matches (25%). YEAST. HEAT SHOCK PROTEIN 26. ( 23748.5 Da) submitted matchedppmstartendPeptide Sequence Modifications 1274.66101274.601746.5200117126(K)DIDIEYHQNK(N)DIDIEYHQNK 1461.85791461.795342.8103176189(K)ADYANGVLTLTVPK(L)ADYANGVLTLTVPK 1729.97191729.901240.8452160175(R)VITLPDYPGVDADNIK(A)VITLPDYPGVDADNIK 1886.09311886.002448.1161159175(K)RVITLPDYPGVDADNIK(A)RVITLPDYPGVDADNIK 12 unmatched masses: 888.3 998.6 1139.6 1211.8 1225.8 1288.7 1314.8 1350.7 1537.3 1788.0 1820.0 2041.1 The matched peptides cover 19% (41/213 AA's) of the protein. In-Gel Digest Hit List : Automated Close External Calibration

37. Sample preparation