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1 Proteomics Informatics Overview of spectrometry (Week 2) Ion Source Analyzer Detector

2 Peptide Fragmentation Ion Source Analyzer 1 Fragmentation Analyzer 2 Detector b y

3 Liquid Chromatography (LC)-MS/MS Ion Source LC Analyzer 1 Fragmentation Analyzer 2 Time Detector

4 Ion Sources Ion Source MALDI ESI Analyzer Detector

5 Electrospray Analyzer Detector

6 Matrix Assisted Laser Desorption Ionization (MALDI) Analyzer Detector V Laser Matrix alpha-cyano-4-hydroxycinnamic acid

7 Detectors Ion Source Analyzer Detector Electron Multiplier Image Current

8 Electron Multiplier Detector

9 Image Current Detector

10 Analyzers Ion Source Analyzer Time-of-Flight Magnetic Sector Quadrupole Ion Trap (3D, linear) FTICR Orbitrap Detector

11 Spectrometry (MS) dv F = ma = m = z( E + v B) dt m dv = E + v B z dt

12 Time-of-Flight Spectrometry zv L = = vt mv 2 2 Ion Source Analyzer Time-of-Flight L Detector Detector t = L 2V m z V

13 Magnetic Sector F = zv = mv R 2 2 mv 2 = zvb R = m z 2V B

14 Quadrupole Filter U V cos( ωt) y x U + V cos( ωt)

15 Ion Trap

16 Fourier transform ion cyclotron resonance F = mv = zvb R 2 m z = R v B = B ω

17 Orbitrap

18 Quadrupole Time-of-Flight Spectrometer Ion Source Analyzer 1 CAD Collision Activated Dissociation Fragmentation Analyzer 2 Detector Quadrupole Time-of-Flight Video

19 Triple Quadrupole Ion Source Analyzer 1 CAD Collision Activated Dissociation Fragmentation Analyzer 2 Quadrupole Quadrupole Quadrupole Detector m/z m/z NO m/z m/z time m/z time YES m/z time time time time m/z m/z YES m/z time time m/z is constant time

20 ESI Linear Ion Trap / Orbitrap Linear Ion Trap HCD Ion Source Analyzer 1 CAD ETD Fragmentation Detector Fragmentation Analyzer 2 Detector Orbitrap Olsen J V et al. Mol Cell Proteomics 2009;8: Video

21 Data Independent Acquisistion 1. MS 2. MS/MS 1 3. MS/MS 2 4. MS/MS 3 5. MS 6. MS/MS 1 7. MS/MS 2 8. MS/MS 3 9. MS 10. MS/MS MS/MS MS/MS MS 14. MS/MS MS/MS MS/MS MS 18. MS/MS MS/MS MS/MS MS 22. MS/MS MS/MS MS/MS 3

22 Data Dependent Acquisistion 1. MS 2. MS/MS 1 3. MS/MS 2 4. MS/MS 3 5. MS/MS 4 6. MS/MS 5 7. MS/MS 6 8. MS/MS 7 9. MS/MS MS/MS MS/MS MS 13. MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS 10

23 Vacuum System Ion Source Vacuum System Analyzer Detector Atmosphere

24 Spectrometry Data Ion Source Analyzer Detector

25 Spectrometry Data Dimensions: Time Peptide m/z Peptide Intensity Petide fragment m/z Peptide fragment...

26 Example data MALDI-TOF m/z Peptide vs m/z m/z

27 Example data ESI-LC-MS/MS Peptide vs m/z vs time m/z Time 100 % Relative Abundance [M+2H] m/z Fragment vs m/z MS/MS

28 Slice - Scalable Data Sharing for Remote Informatics Developed by Manor Askenazi slice.ionomix.com Most mass spectrometry data is acquired in discovery mode, meaning that the data is amenable to open-ended analysis as our understanding of the target biochemistry increases. In this sense, mass spectrometry based discovery work is more akin to an astronomical survey, where the full list of object-types being imaged has not yet been fully elucidated, as opposed to e.g. micro-array work, where the list of probes spotted onto the slide is finite and well understood.

29 Example Spectrometry Data Peptide vs time For m/z which corresponds to 3+ of LGEHNIDVLEGNEQFINAAK

30 Example Spectrometry Data Fragment vs m/z For 3+ of LGEHNIDVLEGNEQFINAAK

31 Example Spectrometry Data Fragment vs m/z For 3+ of LGEHNIDVLEGNEQFINAAK

32 Example Spectrometry Data 3+ of LGEHNIDVLEGNEQFINAAK Peptide vs m/z

33 Example Spectrometry Data 3+ of LGEHNIDVLEGNEQFINAAK Peptide vs m/z

34 Example Spectrometry Data Peptide vs m/z Peptide vs time

35 Openslice.fenyolab.org

36 Proteomics Informatics Overview of spectrometry (Week 2) Ion Source Analyzer Detector

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