PROTEOMICS

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PROTEOMICS
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The omics nomenclature
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A few definitions
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Current -omics
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The proteome is defined as the set of all
expressed proteins in a cell, tissue or organism
(Wilkins et al., 1997).
Proteomics can be defined as the systematic
analysis of proteins for their identity, quantity
and function.
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Proteome Genome
dynamic static
No amplification possible Amplification possible
Hetergenous molecules Homogenous molecules
Large variability of the amount No variability of the amount
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Complexity of the proteome
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Applications of Proteomics

• Mining identification of proteins (catalog the
  proteins)
• Quantitative proteomics defining the relative or
  absolute amount of a protein
• Protein-expression profile identification of
  proteins in a particular state of the organism
• Protein-network mapping protein interactions in
  living systems
• Mapping of protein modifications how and where
  proteins are modified.

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Proteins classes for Analysis

• Membrane
• Soluble proteins
• Organelle-specific
• Chromosome-associated
• Phosphorylated
• Glycosylated
• Multi-protein complexes

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General flow for proteomics analysis
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Current Proteomics Technologies

• Proteome profiling/separation
• 2D SDS PAGE (two-dimensional sodium
  dodecylsulphate polyacrylamide gel
  electrophoresis)
• 2-D LC/LC
• (LC Liquid Chromatography)
• 2-D LC/MS
• (MS Mass spectrometry)
• Protein identification
• Peptide mass fingerprint
• Tandem Mass Spectrometry (MS/MS)
• Quantative proteomics
• - ICAT (isotope-coded affinity tag)
• - SILAC (stable isotopic labeling of amino
  acids)

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2D-PAGE gel
The first dimension (separation by isoelectric
focusing) - gel with an immobilised pH gradient -
electric current causes charged proteins to move
until it reaches the isoelectric point (pH
gradient makes the net charge 0)
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Isoelectric point (pI)

• Separation by charge

Low pH Protein is positively charged
At the isolectric point the protein has no net
charge and therefore no longer migrates in the
electric field.
High pH protein is negatively charged
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2D-SDS PAGE gel
The first dimension (separation by isoelectric
focusing) - gel with an immobilised pH gradient -
electric current causes charged proteins to move
until it reaches the isoelectric point (pH
gradient makes the net charge 0)
The second dimension (separation by mass) -pH
gel strip is loaded onto a SDS gel -SDS denatures
and linearises the protein (to make movement
solely dependent on mass, not shape)
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2D-SDS PAGE gel
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2D-gel technique example
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Some limitations of 2DE

• Limited dynamic range of detection - bias towards
  high abundant proteins
• Co-migration of proteins
• Separation of proteins
• Basic proteins (IP gt 10)
• Hydrophobic proteins
• Small and large proteins (lt 10 gt150 kDa)

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Methods for protein identification
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Mass Spectrometry (MS) Stages

• Introduce sample to the instrument
• Generate ions in the gas phase
• Separate ions on the basis of differences in m/z
  with a mass analyzer
• Detect ions

Vacuum System
Samples HPLC
MALDI ESI
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Mass spectrometers used in proteomic research
Aebersold, R. and Mann, M. (2003) Mass
spectrometry-based proteomics. Nature, 422,
198-207.
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Principles of MALDI-TOF Mass Spectrometry
Mann, M., Hendrickson, R.C. and Pandey, A. (2001)
Analysis of proteins and proteomes by mass
spectrometry. Annu Rev Biochem, 70, 437-473.
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Electro-spray ionisation
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Methods for protein identification
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Protein identification by Peptide Mass fingerprint

• Use MS to measure the masses of proteolytic
  peptide fragments.
• Identification is done by matching the measured
  peptide masses to corresponding peptide masses
  from protein or nucleotide sequence databases.

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Mass spectometry (MS)
Mass spectrometry method of separating
molecules based on mass/charge ratio
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Protein Identification by MS
Spectrum of fragments generated
MATCH
Library
Database of sequences (i.e. SwissProt)
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MALDI peptide map and identification of a
protein. A 116-kDa band was excised and subjected
to tryptic digestion in gel.
Mann, M., Hendrickson, R.C. and Pandey, A. (2001)
Analysis of proteins and proteomes by mass
spectrometry. Annu Rev Biochem, 70, 437-473.
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Advantages vs. Disadvantages

• Ambiguous results difficult to interpret
• Requires sequence databases for analysis

• Determination of MW
• High-throughput capability
• Relative low costs

Limitations can be overcome by peptide sequencing
using tandem mass spectrometry
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How the protein sequencing works?

• Use Tandem MS two mass analyzer in series with a
  collision cell in between
• Collision cell a region where the ions collide
  with a gas (He, Ne, Ar) resulting in
  fragmentation of the ion
• Fragmentation of the peptides in the collision
  cell occur in a predictable fashion, mainly at
  the peptide bonds (also phosphoester bonds)
• The resulting daughter ions have masses that are
  consistent with known molecular weights of
  dipeptides, tripeptides, tetrapeptides

Ser-Glu-Leu-Ile-Arg-Trp
Collision Cell
Ser-Glu-Leu-Ile-Arg
Ser-Glu-Leu-Ile
Ser-Glu-Leu
Etc
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Peng, J. and Gygi, S.P. (2001) Proteomics the
move to mixtures. J Mass Spectrom, 36, 1083-1091.
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Schematic of a quadrupole TOF instrument
After traversing a countercurrent gas stream
(curtain gas), the ions enter the vacuum system
and are focused into the first quadrupole section
(q0). They can be mass-separated in Q1 and
dissociated in q2. Ions enter the time-of-flight
analyzer through a grid and are pulsed into the
reflector and onto the detector, where they are
recorded. There are 14,000 pulsing events per
second. Mann, M., Hendrickson, R.C. and Pandey,
A. (2001) Analysis of proteins and proteomes by
mass spectrometry. Annu Rev Biochem, 70, 437-473.
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Peptide Fragmentation
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Tandem Mass Spectrometry
Isolates individual peptide fragments for 2nd
mass spec can obtain peptide sequence
(trypsin)
Compare peptide sequence with protein databases
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Advantages vs. Disadvantages

• High capital costs
• Requires sequence databases for analysis

• Determination of MW and aa. Sequence
• Detection of posttranslational modifications
• High-throughput capability

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Coupling of LC and tandem MS
Tryptic digested proteins
LC
Ion trap MS
75 µm RP

• Peptide
• MW
• Sequence
• Modification

200 nL to MS
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Reverse Phase column
Polypeptides enter the column in the mobile
phase the hydrophobic foot of the
polypeptides adsorb to the hydrophobic (non
polar) surface of the reverse-phase material
(stationary phase) where they remain until the
organic modifier concentration rises to critical
concentration and desorbs the polypeptides
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Data acquired - Chromatogram
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Triple Play
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Triple Play Dynamic Exclusion
Scan 4502
Scan 4503
2
Scan 4501
626.3
100
95
90
85
1
80
75
70
65
60
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c Full ms 400.00-2000.00
Relative Abundance
50
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835.5
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982.4
35
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610.2
1054.4
25
1156.2
852.2
20
1157.5
703.2
885.0
578.8
503.9
765.9
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1217.7
445.1
1469.7
1259.8
10
5
0
400
600
800
1000
1200
1400
1600
1800
2000
m/z
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Triple Play Dynamic Exclusion
721.2
100
95
Scan 4506
90
85
Scan 4505
80
3
c d Full ms2 852.26_at_35.00 220.00-2000.00
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Scan 4504
70
65
60
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471.0
50
45
40
1261.0
35
30
697.1
25
636.8
1141.9
1
1076.2
20
787.5
611.5
1029.1
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1558.2
515.2
340.0
830.0
1648.0
930.3
10
5
0
400
600
800
1000
1200
1400
1600
1800
2000
m/z
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2D - LC/MS
Peng, J. and Gygi, S.P. (2001) Proteomics the
move to mixtures. J Mass Spectrom, 36, 1083-1091.
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Multidimensional Protein Identification
Technology (MudPIT).
Whitelegge JP (2002) Plant proteomics BLASTing
out of a MudPIT. Proc Natl Acad Sci U S A 99
11564-6.
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Koller A, Washburn MP, Lange BM, Andon NL, Deciu
C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei
J, Wolters D, Yates JR, 3rd (2002) Proteomic
survey of metabolic pathways in rice. Proc Natl
Acad Sci U S A 5 5.
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