proteomics. composition – splice variants – post translational modifications interactions...

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

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700

800

-8 -6 -4 -2 0 2 4 6 8 10

Log(Ratio) base 2

Log2ratio

Fre

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