1. Proteomics Informatics – Overview of Mass spectrometry (Week 2)
Ion Source
Mass Analyzer
Detector
intensity
mass/charge

2. Peptide Fragmentation
Mass Analyzer 1
Frag-mentation
Detector
Ion Source
Mass Analyzer 2 b y

3. Liquid Chromatography (LC)-MS/MS
Ion Source
Mass Analyzer 1
Frag-mentation
Mass Analyzer 2
Detector
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
intensity
mass/charge
Time

4. Ion Sources Ion Source Mass Analyzer Detector intensity mass/charge
MALDI
ESI
intensity
mass/charge

5. Electrospray
Mass Analyzer
Detector

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

7. Detectors Ion Source Mass Analyzer Detector intensity mass/charge
Electron Multiplier
Image Current
intensity
mass/charge

8. Electron Multiplier Detector

9. Image Current Detector

10. Mass Analyzers Ion Source Mass Analyzer Detector intensity mass/charge
Time-of-Flight
Magnetic Sector
Quadrupole
Ion Trap (3D, linear)
FTICR
Orbitrap
intensity
mass/charge

11. Mass Spectrometry (MS)

12. Time-of-Flight Mass Spectrometry
Ion Source
Mass Analyzer
Detector
Time-of-Flight
Detector
Detector
Ion mirror

13. Magnetic Sector

14. Quadrupole Mass Filtery x

15. Ion Trap

16. Fourier transform ion cyclotron resonance

17. Orbitrap

18. Quadrupole Time-of-Flight Mass Spectrometer
Ion Source
Detector
CAD – Collision
Activated
Dissociation
Mass Analyzer 1
Frag-mentation
Mass Analyzer 2
Quadrupole
Time-of-Flight

19. Triple Quadrupole Ion Source Detector Mass Analyzer 1 Frag-mentation
CAD – Collision
Activated
Dissociation
Mass Analyzer 1
Frag-mentation
Mass Analyzer 2
Quadrupole
Quadrupole
Quadrupole
m/z
m/z NO
m/z
time
time
time
intensity
m/z
m/z
YES
m/z
time
time
mass/charge
time
m/z
m/z
YES
m/z
time
time
time
Dm/z is constant

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

21. Data Independent Acquisistion
1. MS
2. MS/MS 1
3. MS/MS 2
4. MS/MS 3
intensity
5. MS
6. MS/MS 1
7. MS/MS 2
8. MS/MS 3
mass/charge
intensity
9. MS
10. MS/MS 1
11. MS/MS 2
12. MS/MS 3
mass/charge
intensity
13. MS
14. MS/MS 1
15. MS/MS 2
16. MS/MS 3
mass/charge
intensity
17. MS
18. MS/MS 1
19. MS/MS 2
20. MS/MS 3
mass/charge
intensity
21. MS
22. MS/MS 1
23. MS/MS 2
24. MS/MS 3 …
mass/charge
intensity

22. Data Dependent AcquisistionMS
MS/MS 1
MS/MS 2
MS/MS 3
MS/MS 4
MS/MS 5
MS/MS 6
MS/MS 7
MS/MS 8
MS/MS 9
MS/MS 10
12. MS
13. MS/MS 1
14. MS/MS 2
15. MS/MS 3
16. MS/MS 4
17. MS/MS 5
18. MS/MS 6
19. MS/MS 7
20. MS/MS 8
21. MS/MS 9
22. MS/MS 10 …
intensity
mass/charge
mass/charge
intensity

23. Vacuum System Ion Source Mass Analyzer Detector Vacuum System
Atmosphere

24. Mass Spectrometry Data
Ion Source
Mass Analyzer
Detector
intensity
mass/charge

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

26. Example data – MALDI-TOF Peptide intensity vs m/z

27. Example data – ESI-LC-MS/MS
Peptide intensity vs m/z vs time
m/z
m/z
% Relative Abundance
100
250
500
750
1000
[M+2H]2+
762
260
389
504
633
875
292
405
534
907
1020
663
778
1080
1022
MS/MS
Fragment intensity vs m/z
Time

28. Charge-State Distributions
MALDI
ESI 1+ 2+ 3+
Peptide
intensity
intensity 4+ 2+ 1+
mass/charge
mass/charge
M - molecular mass
n - number of charges
H – mass of a proton
MALDI
ESI 2+
27+ 3+ 1+
Protein
31+
intensity 4+
intensity 5+
mass/charge
mass/charge

29. Charge-State Example:
M - molecular mass
n - number of charges
H – mass of a proton
Example:
peptide of mass 898 carrying 1 H+ = ( ) / 1 = 899 m/z
carrying 2 H+ = ( ) / 2 = 450 m/z
carrying 3 H+ = ( ) / 3 = m/z

30. Isotope Distributions
12C
14N
16O 1H
32S
+1Da
Intensity
+2Da
+3Da
m/z
m/z
m/z
0.015% 2H
1.11% 13C
0.366% 15N
0.038% 17O, 0.200% 18O,
0.75% 33S, 4.21% 34S, 0.02% 36S
Only 12C and 13C:
p=0.0111
n is the number of C in the peptide
m is the number of 13C in the peptide
Tm is the relative intensity of
the peptide m 13C
𝑇 𝑚 = 𝑛 𝑚 𝑝 𝑚 (1−𝑝) 𝑛−𝑚

31. Isotope distributions
Intensity ratio
Intensity ratio
Peptide mass
Peptide mass
GFP 29kDa
monoisotopic
mass
m/z

32. Resolution M = full width at half maximum (FWHM)
= resolving power M
Resolution = minimum peak separation, M,
which allows to distinguish two ion species
M = full width at half maximum (FWHM)
500
Relative Intensity
50 % I I I I I
499.5
500.0
500.5
501.5
502.0
m/z
Resolution = M/M = 500/0.5 = 1000

33. Resolution

34. Resolution M
R =
= resolving power M
What resolution do we need to differentiate a 1600 Da peptide that carries either an acetylation ( ) or trimethylation ( )?
R = 1600/ = 43,956

35. Isotope Clusters and Charge State1+ 1
Intensity
m/z 2+
0.5
Intensity
m/z 3+
0.33
Intensity
m/z

36. Isotope Clusters and Charge State
Possible to
Determine Charge?
Yes
Maybe No
Intensity
m/z

37. What is the Charge State?
between the isotopes is 0.5 Da
between the isotopes is 0.33 Da

38. Slice - Scalable Data Sharing for Remote Mass 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.

39. Example Mass Spectrometry Data Peptide intensity vs time
For m/z which corresponds to 3+ of LGEHNIDVLEGNEQFINAAK

40. Example Mass Spectrometry Data Fragment intensity vs m/z
For 3+ of LGEHNIDVLEGNEQFINAAK

41. Example Mass Spectrometry Data Fragment intensity vs m/z
For 3+ of LGEHNIDVLEGNEQFINAAK

42. Example Mass Spectrometry Data Peptide intensity vs m/z
3+ of LGEHNIDVLEGNEQFINAAK
Peptide intensity vs m/z

43. Example Mass Spectrometry Data Peptide intensity vs m/z
3+ of LGEHNIDVLEGNEQFINAAK
Peptide intensity vs m/z

44. Example Mass Spectrometry Data
Peptide intensity vs m/z
Peptide intensity vs time

45. Homework: Explore Mass Spectrometry Data
Openslice.fenyolab.org

46. Proteomics Informatics – Overview of Mass spectrometry (Week 2)
Ion Source
Mass Analyzer
Detector
intensity
mass/charge