1. Proteomics Global representation of protein
composition, interactions, modifications , and
activity in temporal context
“High content” implicit

2. Approaches High resolution separation Arrays Multiplex
SDS PAGE 1-2D
IEF
Multidimensional LC
Arrays
Protein
Antibody
Multiplex
Luminex
Aptamers
Mass spectrometry

3. MS based proteomic work flow
Sample
Ionize
Determine mass
Process
Process Ions
Process Data

4. Ionization methods for peptides
Electrospray ionization (ESI) Matrix assisted laser desorption ionization (MALDI)

5. Beam mass analysers
2:140 (2003)

6. Trapping mass analysers
Quadrupole ion trap
Fourier transform ion cyclotron resonance

7. Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware implementations.
Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware implementations. A, the stacked ring ion guide (S-Lens) increases the ion flux from the electrospray ion source into the instrument by a factor 5–10. B, the dual linear ion trap design enables efficient trapping and activation in the high-pressure cell (left) and fast scanning and detection in the low pressure cell (right). C, the combo C-trap and HCD collision cell with an applied axial field with improved fragment ion extraction and trapping capabilities.
Olsen J V et al. Mol Cell Proteomics 2009;8:
©2009 by American Society for Biochemistry and Molecular Biology

8. QqTOF Layout

9. Peptide Fragmentation
Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010

10. Archives of Physiology and Biochemistry, 2009; 115(5): 311–319

11. Tandem Mass Spectrometry (MSMS)
Molecular Systems Biology :222

12. Proteins: Considerations
Virtually any source
Free from degradation
Minimal unintroduced chemical modifications
Quantity a few micrograms
Concentration mg/ml
Free from inhibitory materials (e.g. SDS, salts)
Dynamic range of materials

13. Separation Approaches
Separation methods
1-2 D PAGE
Multidimensional LC
Capillary electrophoresis
Affinity
Antibody
Lectin
Substrate cofactor
PTM

14. Gel separated Proteins

15. 2D SDS PAGE Strengths Limitations fair resolution Meta data Load
size and isoelectric point
Molecular heterogeneity (PTMs)
Limitations
Load
Solubility
Isoelectric point extremes
molecular size
variability

16. 2D Difference Gel electrophoresis (DIGE)
AppliedBiomics.com

17. Nat. Inst. Aging

18. iTRAQ—Isobaric Tags for Relative and Absolute Quantification
Multiple tag types available from
different commercial sources.
Numbers tags/kit constantly rising.
Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010

19. Comparison of SILAC and iTRAQ
Basis of labelling
Biosynthetic
Chemical
Sample labelling
Simple
Labour intensive
Requirements
Active metabolism
Proteins
Defined media
Multiplexing
Yes
Signal
Diluted
Single ID
Inference some times
Confirmed
Potential for Bias
low
higher
Reutilisation
Not applicable
Cellular capacity

20. Induction of Podocalyxin in EMT
Log2ratio
Frequency
Control TGF Beta
Control
+ TGFβ1
- TGFβ1
72h
Combine
SDS PAGE
LC MS/MS
TGF

21. PTM Analysis
14:35 (2013)

22. Selected Reaction Monitoring
Targeted for selected analytes.
Not a discovery approach for protein identification
Increases sensitivity fold
Qualitative or quantitative
Offers multiplexing capacity analytes per run

23. Selected Reaction Monitoring
When antibodies not available
Because targeted can ignore high abundance
Potential to detect/quantify multiple analytes
Demonstrated in this case off by several hundred fold because of interfering antibodies
Validation
20-30 different proteins simultaneously in a single sample
Used in RA, Renal tspt, biofuels effects of substrate, IP analysis

26. Activity based protein profiling
Inactive enzyme
Active enzyme
Labelled
Unlabelled
Carboxyl group of acid hydrogen bonds with histidine - makes electron pair more electronegative
Probes available for many categories of enzymes e.g.
serine hydrolase, cysteine proteases, ubiquitinase

27. Marker 2E4A-SH 2E4B-SH LG8A-SH LG8B-SH Normal Lethal
Nan Li , Herman S Overkleeft , Bogdan I Florea
Activity-based protein profiling: an enabling technology in chemical biology research
Current Opinion in Chemical Biology 16:

28. 2.55 Å crystal structure of SOMAmer SL1025 bound to human IL-6 (form 2 chains A and B).
2.55 Å crystal structure of SOMAmer SL1025 bound to human IL-6 (form 2 chains A and B).A, helices of IL-6 are labeled (A–D) from N to C terminus (term) and are colored magenta (helix A), purple (helix B), blue (helix C), and cyan (helix D). The modified nucleotides of the SOMAmer are colored gold. This color scheme is maintained throughout the figures. B, chemical structures of the C-5 modified deoxyuridines present in SOMAmer SL1025.
Gelinas A D et al. J. Biol. Chem. 2014;289:
©2014 by American Society for Biochemistry and Molecular Biology