Proteomic based approaches to biological problems

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Proteomic based approaches to biological problems

• Toni Slabas

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How is biology organised?orHow do living
organisms function
Aristotle
Lucretius

• Whole genomes have been sequenced
• What is the importance /function of genes
• Can we learn all from the DNA/transcript level?

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Molecular Relationships
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mRNA via DNA Array
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Iyer VR et al. (1999) The transcriptional program
in the response of human fibroblasts to serum.
Science 283 83-87
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Unsync 24 hr 20 hr 16 hr 12 hr 8
hr 6 hr 4 hr 2 hr 1 hr 30
min 15 min 0 hr
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Can you ascribe gene function?
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Lactate dehydrogenase
e-crystallin
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PROTEOMICS
PROTEOLYTIC PROCESSING POLYPEPTIDE
SPLICING N- TERMINAL MET ACETYLATION ,
PANTHETHENYLATION MYRISTOYLATION FARNESYLATION GLY
COSYL-PHOSPHATIDYLINOSITOL SULPHATION HYDROXYLATIO
N PHOSPHORYLATION DISULPHIDE BOND FORMATION

• ACTIVITY
• LOCATION
• TURNOVER

BIOLOGICAL FUNCTION
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Post-translational modifications
Pre-protein
C-terminus
N-terminus
signal peptide
cleavage
Mature protein
Modified protein
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Events are dynamic and protein states change
What can you see?

Calyculin A Phosphothreonine State of
phosphorylation Rapid and slow events
Protein phosphatase inhibitor
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Phospho-Threonine
Untreated
Calyculin A
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Protein World
Dynamic
Size 9KDa- 200KDa pI -acidic/basic Covalent
modifications Hydrophobicity
Catalytic, structural, regulatory
Complexes
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Arabidopsis
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Plant Cell
Cell wall fragments
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Sensitivity, reliability and resolution
Efraim Racker

• Dont waste clean thoughts on dirty enzymes!

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Technology

• Importance/ detection and assignment
• Coverage of protein sequences via mass
  assignments.
• Ionizations and MALDI-TOF . M Sensitivity
• MS-MS M2 y-ions and b-ions
• Gel based methods and limitations - 2D-DIGE
• Complex separation and ICAT
• SILAC
• iTRAQ

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Detection and assignment

• MALDI-TOF - gives predicted
• peptides masses at 10ppm
• accuracy
• MS-MS Sequencing - de novo
• sequencing
• Data base requirements

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MALDI-TOF mass spectrometry of peptides
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Parent protein Mr 11,800 pI 3.7
K
K
K
R
R
NH2
CO2H
Trypsin
Tryptic peptides
K
K
R
K
R
NH2
(3)
(4)
(6)
CO2H
(5)
(1)
(2)
a-cyano-4-hydroxy cinnamic acid (ionisation
matrix)
LASER
MALDI-TOF
Spectrum
Peptide Masses (Da) (1) 900 (2) 1300 (3) 1700 (4)
2300 (5) 2500 (6) 3100
2300
2500
Relative Intensity
3100
1300
900
Identification
Mass/charge
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MALDI-TOF Spectrum
Mass
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MOWSE Score - probability based on mass accuracy
plus coverage
Pappin DJC et al., 1993 Current Biology
3327-332 MOlecular WEight SEarch A matching of
as few as 3-4 peptides is sufficient to uniquely
identify a protein in a data base of 50,000
proteins
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Peptide coverage via MALDI-TOF oleate
hydroxylase
1 MGGGGRMSTV ITSNNSEKKG GSSHLKRAPH TKPPFTLGDL
KRAIPPHCFE RSFVRSFSYV 61 AYDVCLSFLF YSIATNFFPY
ISSPLSYVAW LVYWLFQGCI LTGLWVIGHE CGHHAFSEYQ 121
LADDIVGLIV HSALLVPYFS WKYSHRRHHS NIGSLERDEV
FVPKSKSKIS WYSKYSNNPP 181 GRVLTLAATL LLGWPLYLAF
NVSGRPYDRF ACHYDPYGPI FSERERLQIY IADLGIFATT 241
FVLYQATMAK GLAWVMRIYG VPLLIVNCFL VMITYLQHTH
PAIPRYGSSE WDWLRGAMVT 301 VDRDYGVLNK VFHNIADTHV
AHHLFATVPH YHAMEATKAI KPIMGEYYRY DGTPFYKALW 361
REAKECLFVE PDEGAPTQGV FWYRNKY
Electrophoretic separation of proteins
In gel digestion trypsin
Elution and clean-up of peptides
Application to MALDI sample grid
Mass spec. analysis and protein identification
Jensen ON et al., (1997) Identification of the
components of simple protein mixtures by
high-accuracy peptide mass mapping and database
searching. Analytical Biochemistry 69 4741-4750
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Peak height is not related to quantity of peptide

• Can get ion-supression
• Arginine -containing peptides dominate the
  spectrum
• Can convert Lys to pseudo-Arg
• Hale JE et al (2000) Anal. Biochem. 287, 110-117

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Tandem mass spectrometry analysis

• Isolation of ions based on m /z
• Fragmentation of ion
• Second mass analysis to get structural
  information

Sequencing of peptide ions
Linked to Electrospray ionisation system
Compatible with HPLC solvent systems Peptide
separation and delivery to mass
spectrometer Very slow flow rates nL per minute
increase sensitivity Nanospray sample delivery
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Sequencing of peptide ions

• Analysis of peptide ions and identification of
  multiply charged ions
• Selection of ions and entry into collision cell
  for fragmentation
• Analysis of product ion spectra peptide
  sequence analysis

Rapid switching between the modes - milliseconds
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Comparing the proteins in biological samples?
- experimental variation - biological variation
Getting quantitative data from older 2D
technology is difficult
2D-DIGE - Two-dimensional differential gel
electrophoresis
Tonge R et al (2001) Validation and development
of fluorescence two-dimensional differential gel
electrophoresis proteomic technology Proteomics 1
377-396
Fluorescent cyanine dyes that react with amino
groups C2, Cy3, Cy5 - charge and
mass-matched - non overlapping
excitation and emission wavelengths
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Silver stained 2-D protein gel of yeast total
cell lysate
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Developing Cy3
Germinating Cy5 Dual ImageCy3/Cy5
E. Coli forming inclusion bodies
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MudPIT

• Multidimesional protein identification technology
• Wu et al (2003) Nature Biotechnology 21, 532-537

Simplification of protein mixture
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Mass spectrometry is not quantitative cannot
directly compare two samples differences in
ionization between different analyses
Specialized preparation and labelling of samples
required before MS anlaysis
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Isotope-Coded Affinity TagsICAT TM
linker (heavy or light)
reactive group
biotin tag
O
X
X
NH
O
HN
O
X
X
I
O
N H
N H
O
X
X
S
X
X
heavy reagent d8-ICAT (Xdeuterium) light
reagent d0-ICAT (Xhydrogen)
S H
cys
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ICAT Approach to Quantitative Protein Expression
Analysis
Determine differences in protein expression by
measuring relative intensities of light vs. heavy
MS
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Light
Heavy
Quantitation
Optional fractionation
ICAT Reagent-labeled cysteines
0
550
560
570
580
m/z
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NH2-EACDPLR-COOH
Affinity separation
Digest
MS/MS
Mixture 2
Identification
0
200
400
600
800
m/z
MDLC or MDLC with 1D gel
Quantitation and protein identification
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iTRAQ tandem MS-MS analysis provides both
identification and relative quantification
between different samples
Ross PL et al., (2004) Multiplexed protein
quantitation in Saccharomyces cerevisiae using
amine-reactive isobaric tagging reagents.
Molecular and cellular proteomics 3.121154-1169
Gels are not required Samples are digested with
trypsin and derivatized with one of
four reagents
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SILAC Stable isotope labelling by amino acids
in cell culture
Growth of cells in media containing essential
amino acid In one sample, the amino acid is
isotopically labelled eg deuterium labelled
L-leucine (leucine d3)
Ong et al., (2002) Stable isotope labelling by
amino acids in cell culture, SILAC as a simple
and accurate approach to expression
proteomics. Molecular and Cellular Proteomics 1.5
376-386
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Incorporation of Leu-d3 with time NIH 3T3 cells
Triply charged peptide VAPEEHPVLLTEAPLNPK
SILAC differentially labels 50 of peptides
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After mixing, proteins and peptides can be
analysed in any of the ways In which they are
analyzed in non-quantitative proteomics
Differentiation of muscle cells 1D
electrophoresis followed by nanospray MS/MS
analysis
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Data sets and what to do with them?

• Does transcriptome data need to align with
  proteome data and what confidence do we have in
  the data?
• Moving towards the next questions?
• What literature do you need to read?

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Examples which have worked

• Park et al - extracellular matrix
• Chivasa - extracellular ATP
• Components of complexes. Vesicle trafficking.
• How many new questions does it open up?

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Systems Biology

• What does it mean?
• Analytical machine or brain.
• Data sets are important but no substitute for
  thinking?

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Arabidopsis
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Figure 1.
1.
IgG
IgG binding
Wash
TEV site
2.
Protease digestion
4.
EGTA elution
3.
Calmodulin binding (plus Ca2)
Identification of constituents
wash
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I know it will be important
One day you will be able to put a tax on it!
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