Proteomics
Global representation of protein
composition, interactions, modifications , and
activity in temporal context
“High content” implicit
Approaches
• High resolution separation
– SDS PAGE 1-2D
– IEF
– Multidimensional LC
• Arrays
– Protein
– Antibody
• Multiplex
– Luminex
– Aptamers
• Mass spectrometry
MS based proteomic work flow
MS
Sample
Process
Ionize
Determine mass
Process Ions
Process Data
Ionization methods for peptides
Electrospray ionization (ESI)
Matrix assisted laser desorption ionization (MALDI)
Beam mass analysers
2:140 (2003)
Trapping mass analysers
a) Quadrupole ion trap
b) Fourier transform ion cyclotron resonance
Schematic of the LTQ Orbitrap Velos MS instrument with three new hardware
implementations.
Olsen J V et al. Mol Cell Proteomics 2009;8:2759-2769
©2009 by American Society for Biochemistry and Molecular Biology
QqTOF Layout
Peptide Fragmentation
Adv Protein Chemistry & Structural
Biology, Vol. 80, 1-44, 2010
Archives of Physiology and Biochemistry, 2009;
115(5): 311–319
Tandem Mass Spectrometry (MSMS)
Molecular Systems Biology 2008 4:222
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
Separation Approaches
• Separation methods
– 1-2 D PAGE
– Multidimensional LC
– Capillary electrophoresis
– Affinity
•
•
•
•
Antibody
Lectin
Substrate cofactor
PTM
Gel separated Proteins
2D SDS PAGE
• Strengths
– fair resolution
– Meta data
• size and isoelectric point
• Molecular heterogeneity (PTMs)
• Limitations
–
–
–
–
–
Load
Solubility
Isoelectric point extremes
molecular size
variability
2D Difference Gel electrophoresis (DIGE)
AppliedBiomics.com
Nat. Inst. Aging
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
Comparison of SILAC and iTRAQ
SILAC
iTRAQ
Basis of labelling
Biosynthetic
Chemical
Sample labelling
Simple
Labour intensive
Requirements
Active metabolism
Proteins
Defined media
Proteins
Multiplexing
Yes
Yes
Signal
Diluted
Single
ID
Inference some times
Confirmed
Potential for Bias
low
higher
Reutilisation
Not applicable
Cellular capacity
Not applicable
Induction of Podocalyxin in EMT
Frequency
Frequency
800
700
600
500
400
300
200
100
0
-8
-6
-4
-2
0
2
4
6
8
10
Log(Ratio) base 2
Log2Control
ratio
Control TGF Beta
- TGFβ1
TGF
72h
+ TGFβ1
Combine
SDS PAGE
LC MS/MS
PTM Analysis
14:35 (2013)
Selected Reaction Monitoring
• Targeted for selected analytes.
• Not a discovery approach for protein
identification
• Increases sensitivity 30-50 fold
• Qualitative or quantitative
• Offers multiplexing capacity 20-30 analytes
per run
Selected Reaction Monitoring
Activity based protein profiling
Unlabelled
Inactive enzyme
Labelled
Active enzyme
Probes available for many categories of enzymes e.g.
serine hydrolase, cysteine proteases, ubiquitinase
Normal
Nan Li , Herman S Overkleeft , Bogdan I Florea
Activity-based protein profiling: an enabling technology in chemical biology research
Current Opinion in Chemical Biology 16:227 2012
Lethal
2.55 Å crystal structure of SOMAmer SL1025 bound to human IL-6 (form 2 chains A and B).
Gelinas A D et al. J. Biol. Chem. 2014;289:8720-8734
©2014 by American Society for Biochemistry and Molecular Biology