1. Protein/Peptide Quantification
An Introduction to Quantification by
Isotope dilution
Chemical labelling
Biological labelling
Advanced scanning methods

2. General SILAC Workflow

3. Quantification by Isotope Comparison

4. Complications are possible

5. Phosphopeptide analysis in the mitotic cell cycle

6. Complications are possible

7. Dynamic SILAC

8. Relative Isotope Abundance
The variance is shown for two proteins
The number of peptides vary but not the ratio

9. Decay curve fitting
The curves all appear to fit a single exponential

10. Variation of decay
Wide variation in decay rates: But why?

11. Multiplexing SILAC
Label with 12C4, 13C4, 13C6, 13C6N4, 13C615N42H7

12. D does not affect elution

13. Western blot support

14. Chemical Labelling: iTRAQ
9.0
436.2
863.4
1290.6
1717.8
2145.0 20
114
115
116
117
Mass (m/z) 40 60 80
100
Isobaric Tag
(Total mass 145 Da)
Reporter
(Mass )
Balance
(Mass )
Peptide Reactive Group
Charged
Neutral Loss
Amine Specific

15. iTRAQ labelling

16. iTRAQ reporter ions

17. iTRAQ 8-plex analysis

18. TMT - Isobaric Mass Tags
Tandem Mass Tags
A family of amine reactive isobaric MS/MS tags based on an identical chemical structure
Six-plex profiling
Tandem Mass Tags (TMT) are an intelligent way to obtain dramatically improved quantitation data allowing for peptide andprotein biomarker discovery, validation, and the development of multiplex assays for routine measurement with analytical performance equivalent to ELISA.
Tandem Mass Tags belong to the family of reagents known as isobaric mass tags. As the term suggests, several different forms of a molecule have the same mass and can be independently attached to a peptide or protein via a reactive group. Two core elements the Mass Reporter (Tag) and the Mass Normaliser(MN) are connected via a Cleavable Linker, which has the tendency to break preferentially during MS/MS fragmentation conditions. By selective placement of isotopes such as 13C, 15N or 18O at certain positions of the Tagand the Mass Normaliser, the mass of the Tag can be varied to produce series of Mass Reporters, typically in 1 Dalton increments, while keeping the mass of the entire label constant.

19. Peptide identification and quantification
Peptide sequence information
Slide courtesy of Atul Deshpande and Travis LaFavor at Pierce

20. General TMT Workflow

21. AQUA - Absolute Quantitation of Peptides
Must use internal standard
Isotopically stable heavy peptides
Rules for selection of a suitable (proteotypic) peptide
Unique to the protein to be quantified
Previously detected by mass spectrometry
Preferred charge state 2+
Avoid peptides with Cys, Met residues
Phosphopeptides can be used for monitoring phosphorylation events

22. AQUA Peptide Workflow Identify a proteotypic peptide
Leu 13C6
Synthesize a heavy peptide
Spiking and LC/MS analysis
Quantitation from full scan MS

23. How a Mass Spectrometer works.......
The ion source is the part of the mass spectrometer that ionizes the material under analysis (the analyte). The ions are then transported by magnetic and electric fields into the mass spectrometer. Ionisation should be a gentle as possible to ensure that an intact molecule is introduced into the mass spectrometer with the minimum of fragmentation. If a molecule is not charged, it cannot be directed by the magnetic or electric fields and will not reach the detector and will therefore not be seen.

24. Triple Quadrupole Scanning Modes
All peptides measured intact Q1 Q2 Q3
m/z
RIC

25. Product ion Scanning Mode (MS/MS)
Only Green is selected for fragmentation Q1 Q2 Q3
m/z
RIC

26. Parent Ion Scanning Mode
All peptides sequentially fragmented
Only parents of selected fragment detected Q1 Q2 Q3
m/z
RIC

27. Searching for PhosphoPeptides

28. Parent ion triggered MS/MS

29. Selective GlycoPeptide Detection (HexNAc)

30. Single Reaction Monitoring
Only peptides with chosen mass fragmented
Only parents of selected fragment detected Q1 Q2 Q3
m/z
RIC

31. Multiple Reaction Monitoring
Only peptides with chosen mass fragmented
Only parents of both selected fragments detected Q1 Q2 Q3
m/z
RIC

32. MS/MS spectrum of the peptide LDVDQALDR

33. TIC of 16 MRM transitions for peptides 1-4
Endogenous and Isotopically labeled pairs

34. Replacing Western Blotting?
Mutant
Gene Name
Western
MRM heavy/light

35. Conclusions Proteomics is slowly becoming useful
Up to 4000 proteins can be identified in a cell
Dynamic range is around 100 and still limited
MRM increases dynamic range to ca 5000
Quantification becoming possible but problematic