Proteomics
Dec 16, 2015
Proteomics
Proteomics
• Composition– Splice variants– Post translational modifications
• Interactions • Distribution• Activity• Dynamics • High content information
TOP DOWN VS BOTTOM UP
Mass Spectrometry
Ionization methods for peptides
Electrospray ionization (ESI) Matrix assisted laser desorption ionization (MALDI)
Tandem Mass Spectrometry (MSMS)
Molecular Systems Biology 2008 4:222
QqTOF Layout
Ion Trap
Methods in Enz. 402:109
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
Peptide Fragmentation
Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010
Archives of Physiology and Biochemistry, 2009; 115(5): 311–319
Typical work flow
Mass spectrometryProteinsPeptides
From proteome lists to biological impact– tools and strategies for the analysis of large MS data sets
PROTEOMICSVolume 10, Issue 6, pages 1270-1283, 13 JAN 2010 DOI: 10.1002/pmic.200900365http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900365/full#fig1
Proteins: Considerations
• Virtually any source e.g. tissues, fluids, cells, cellular components, supernatants
• Free from degradation. Rapid harvest and processing or storage.
• A few micrograms required for analysis but must be reasonably concentrated
• Free from inhibitory materials• Dynamic range of materials
Compositional Questions
• What proteins are present?• Are their protein differences between two
samples?– Diseased vs normal– Differentiated vs undifferentiated– Resting vs activated– Wild type vs knockout
Separation Approaches
• Separation methods– 1-2 D PAGE– Multidimensional LC– Capillary electrophoresis– Affinity
• Antibody• Lectin• Substrate cofactor• PTM
1and 2D LC
• Orthogonal separations– Protein level in principle not generally in practice– MUDPIT on line– 1st dimension offline
• Many types of separation
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
Biosynthetic Labeling• Based on incorporation of stable isotope labels• Benefits
– Uniform labeling– Minimally manipulative– Samples processed as single batch
• Considerations– Incorporation efficiency
• Division• Turnover rate
– Catabolism– PTMs– Cost – Cell growth in SF or reduced media
Nat. Inst. Aging
Triple SILAC
Proteome Res., Article ASAPDOI: 10.1021/pr200740aOctober 20, 2011
Stable Isotope Labeling
• Dual tags– e,.g. ICAT
• SH specific• Reduces complexity but may limit number of useful
peptides for ID
• Multiplex (up to 10)– ABI iTRAQ– PE ExacTag
iTRAQ—Isobaric Tags for Relative and Absolute Quantification
Adv Protein Chemistry & Structural Biology, Vol. 80, 1-44, 2010
Comparison of SILAC and iTRAQSILAC 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
Phosphoproteomics
Proteomics 2007, 7, 2751
Isolation methods
Proteomics 2007, 7, 2751
Derivatization methods
Comparison of phosphorylation enrichment methods
Proteomics 2007, 7, 2751
TCR Activation specific phospho sites
Sci. Signal. 2009 Vol. 2, p. ra46
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 applied to proteomics
Journal of Mass SpectrometryVolume 46, Issue 3, pages 298-312, 10 MAR 2011 DOI: 10.1002/jms.1895http://onlinelibrary.wiley.com/doi/10.1002/jms.1895/full#fig1
Selected Reaction Monitoring
Selected reaction monitoring applied to proteomics
Journal of Mass SpectrometryVolume 46, Issue 3, pages 298-312, 10 MAR 2011 DOI: 10.1002/jms.1895http://onlinelibrary.wiley.com/doi/10.1002/jms.1895/full#fig5
Bioorthogonal Chemistry
Bioorthogonal CLICK Chemistry
Activity Based Protein Profiling
Mechanism of probe action
Inactive enzyme
Active enzyme
Labelled
Unlabelled
Nomura et. alNature Rev. Cancer10:630 (2010)
Marker
2E4A-SH
2E4B-SH
LG8A-SH
LG8B-SH
Normal Lethal
12k17k
24k
31k
38k
52k
76k
102k
150k
225k
Marker
2E4 Cell ly
sate
LG8 Cell ly
sate
2E4-Serine H
ydrolase
LG8-Serine H
ydrolase
12k17k
24k
31k
38k
52k
76k
102k
150k
225k
Marker
2E4 Cell ly
sate
LG8 Cell ly
sate
2E4-Serine H
ydrolase
LG8-Serine H
ydrolase
Commassie
Urine Profiling in Renal Transplant
Summary :Activity based protein profiling
• Adjunct to other high content approaches• Feasible to apply directly to clinical samples for rapid
monitoring and quantitation of activity • Full coverage of activities not attainable at this time• Potential for identification of new processes• Feasibility of point of care analysis in near real time• Potential for arrayed format for multiplexing• Utility in infectious diseases?
Examples
Cell Migration Requires Dynamic and Regional Processes
MW + Lys
What does enrichment Mean?
Candidate Selection
• 119 selected based on “enrichment”• Screened ~90 clones targeting 22 proteins
using shRNAmir mediated silencing • 15 targets with 2 or more clones inhibiting
migration by >50% and outside 3 SD of assay variation.
NK Synapse (NKIS)
IQGAP1 involvement in MTOC and granule polarization in NK cell cytotoxicity‐
European Journal of ImmunologyVolume 41, Issue 9, pages 2763-2773, 3 AUG 2011 DOI: 10.1002/eji.201040444http://onlinelibrary.wiley.com/doi/10.1002/eji.201040444/full#fig9
NK Granule Release
LC MSMS Contents ~400 proteinsMembrane and associated proteins
Acknowledgements
• MCPSB– Namita Kanwar– Dmitry Shanshun– Paymen Ezzati– Peter Nickerson– John Cortens– Xiaobo Meng– Ravi Dwivedi– William Summers– Patty Sauder– Dustin Lippert– Mario Navarrete
• Oleg Krokhin
• Chris Bleackley
• ThermoFisher Pierce– Monica O’Hara– Ryan Bomgarden
Morphological and phenotypic changes associated with EMT
Acloque H et al. J Clin Invest. 2009; 119:1438
i-ii Interactionsii-iii ECM iii Polarityiv Mobility
Control
TGF
Control TGF Beta
SILAC Labelleing
+ TGFβ1
- TGFβ1
72hK and R C12, N14
K and R C13, N15
Combine SDS PAGE
LC MS/MS
Data analysis
• 3500 proteins identified with confidence scores >95%
• Only proteins observed in biological replicates included in current analysis
• Those outside 95% interval of the population (z score >1.96) selected
• 45 up regulated• 36 down regulated
0
100
200
300
400
500
600
700
800
-8 -6 -4 -2 0 2 4 6 8 10
Log(Ratio) base 2
Log2ratio
Fre
quen
cy