Integrating PROTEOMICS into your Functional Genomics Program

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Proteomics
Ref Pandey, A. and M. Mann. Proteomics to study
genes and genomes. Nature 405 837-846 (2000).
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PROTEOMICS

• Large-scale analysis of the expressed proteins
  from the genome.

WHY

• Protein profiling
• Cellular location
• Protein interactions
• Post-translational modifications

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Whole Cell Analysis of Gene Expression
Transcription - ?mRNA
Post-transcription - ?PROTEINS
DNA Microarrays
2-D gels
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Protein Profiling 2-D gel analysis
Mass spectrometric
identification
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Differential Display Proteomics
Vs.
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Why knowing interactions and modifications are
important
1. Identify function of protein Guilt by
association 2. Identify function of
uncharacterized proteins 3. Identify new roles
for characterized proteins 3. Identify
mechanisms that regulated protein activity 4.
Establish networks of protein interactions
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Methods we are using to isolate protein complexes
1. Direct purification

2. Antibodies to native protein
3. Antibodies to epitope-tag(eg. Myc)
4. Affinity purification (eg. GST)
5. Tandem affinity purification
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Proteomic analysis
1-D SDS-PAGE
Isolated Protein Complex
In situ spot digest
Resolve by
2-D gel electrophoresis
Mass spectrometry (MALDI-TOF/ LC-MS/MS)
Database search
Protein identification
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Mass Spectrometry A Brief Definition Mass
Spectrometry is a technique for the production of
charged molecular species in vacuo, and their
separation by magnetic and/or electric fields
based on the mass to charge ratio (m/z).
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Monoisotopic mass the sum of all the lightest
isotopes.
Average mass the abundance weighted sum of all
the isotopes of all the elements present.
Amino acid 3LC SLC Average
Monoisotopic Glycine Gly G
57.0519 57.02146 Alanine Ala A
71.0788 71.03711 Serine
Ser S 87.0782 87.02303 Proline
Pro P 97.1167
97.05276 Valine Val V
99.1326 99.06841 Threonine Thr T
101.1051 101.04768 Cysteine
Cys C 103.1388 103.00919 Leucine
Leu L 113.1594
113.08406 Isoleucine Ile I
113.1594 113.08406 Asparagine Asn
N 114.1038 114.04293 Aspartic acid
Asp D 115.0886 115.02694 Glutamine
Gln Q 128.1307
128.05858 Lysine Lys K
128.1741 128.09496 Glutamic acid Glu
E 129.1155 129.04259 Methionine
Met M 131.1926 131.04049 Histidine
His H 137.1411
137.05891 Phenyalanine Phe F
147.1766 147.06841 Arginine Arg
R 156.1875 156.10111 Tyrosine
Tyr Y 163.1760 163.06333 Tryptophan
Trp W 186.2132 186.07931
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Information mass spectrometry returns
Trypsin digest
MS
MS/MS
975
S
1644
F
G
635
1239
D
I
Y
I
1734
435
m/z
m/z
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Two Ionization Methods for Peptides and Proteins
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Mass Spectrometry of Peptides
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MALDI Target
(3)
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MALDI Time-of-Flight Mass Spectrometer
Laser Optics
Nitrogen Laser (337 nm)
TOF Analyzer
Microchannel Detector
MALDI Target
?
Ion Mirror
Ion Grid
(3)
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Peptide Mapping by MALDI
8000
7000
6000
5000
Intensity (Counts)
4000
3000
2000
1000
500
1000
1500
2000
2500
3000
Mass (m/z)
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Peptide Mapping by MALDI
1394.49
Protein Beta Galactosidase
(from E. Coli) M.W. 116,278
Da Coverage 18.1 (15/17 peptides)
8000
7000
6000
1099.34
1428.44
5000
1067.31
1252.42
Intensity (Counts)
861.24
1742.46
1457.47
1584.48
4000
1361.48
989.30
900.22
750.24
3000
2846.95
1787.43
2522.78
2000
1000
500
1000
1500
2000
2500
3000
Mass (m/z)
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Protein Identification Using Mass Spectral Data
Database Searching.
Free access internet-based search
algorithms (using peptide mass map or
fragmentation data) Matrix Science--
http//www.matrixscience.com/ Mascot Prowl--
http//www.rockefeller.edu/ProFound,
PepFrag Protein Prospector-- http//prospector.u
csf.edu/MS-FIT, MS-TAG ExPAsy--
http//us.expasy.org/tools/peptident.htmlPeptiden
t
Newer version is available recently acquired by
genomic solutions (www.genomicsolutions.com).
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Database Search Considerations
Calibration
Mass tolerance
Isotopic Distribution
Two peptides with 1.04 Da difference in mass (/-
0.027 Da _at_ 20 ppm mass error).
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MASCOT (www.matrixscience.com)
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• Peptide Mapping by MALDI

• Advantages
• Rapid sample preparation and analysis
• Inexpensive
• Public tools available on the Internet
• Large databases are readily available
• Disadvantages
• Requires relatively pure samples
• Signal intensity may be adversely affected
  by buffers, salts and detergents.
• Post-translational modifications may obscure
  results
• Manual data entry and searching is tedious
• Not all genomes have been sequenced
• Sequence coverage may be low because many
  peptides do not yield a MALDI signal

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De Novo Sequencing
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Sequencing a Peptide by Fragmentation
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• Principles of Electrospray Ionization

• Solution-phase Ionization of Peptides and
  Proteins
• Primary ionization takes place in solution
  based on simple acid-base chemistry
• Samples are sprayed into a desolvation chamber
  where the solution is successively evaporated
  using heat, nitrogen drying gas and strong
  electric fields to form sub-micron size
  droplets, which ultimately form desolvated
  charged molecules
• R-NH2 H3O R-NH3
  H2O
• R-CO2H HO- R-CO2-
  H2O

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Electrospray Ion Source
Nebulization Gas (N2)
MS Sample Orifice
Protein/Peptide (0.01 10 mM)
Flow 5 100 mL/min
Ion Current
4 kV
20 V
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Nanospray Ionization
MS Sample Orifice
Protein/Peptide (0.01 10 mM)
Flow 10 50 nL/min
2 - 4 mm Capillary tip
1 kV
20 V
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Liquid Chromatograph - Tandem Mass Spectrometer
Electrospray Ion Source
Detector
Q1
Capillary HPLC
Q3
Collision Cell (Q2)
Electron Multiplier
Ion Source Controller
Analog - RF Scan Controller
Electrometer Amplifier
Instrument Controller
Data Processing Storage
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Using Sequence Databases to Interpret MS/MS Data
-the SEQUEST Algorithm
Compare all possible sequences in database with
mass equal to parent mass.
Parent mass Fragment ion masses.
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SEQUEST PROCESS
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Global Protein Expression Methods via MS
(MS is a universal detector)
in vivo Stable isotope labeling
in vitro Stable isotope labeling
label
State 2
State 1
State 1
State 2
Harvest Cells
Harvest Cells
label
Mix Cells
1 2
State 2
State 1
Purify Proteins
Mix Proteins
Digest Proteins with Trypsin and Analyze by
LC-MS/MS
1 2
Purify Proteins
Digest Proteins with Trypsin and Analyze by
LC-MS/MS
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Isotope Coded Affinity Tag (ICAT) reagents
d0- or d8-ICAT
(X H or D)
LDQWLCEK
100
d0
d8
1520
1525
1530
1535
1540
50
Relative Magnitude
0
800
1200
1600
2000
2400
m/z
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Protein Quantification Identification via ICAT
Strategy
Mixture 1
100
membrane
nuclear
cytosolic
Light
Heavy
Optional fractionation
ICAT-labeled cysteines
0
550
560
570
580
m/z
100
NH2-EACDPLR-COOH
Affinity separation
Combine, proteolyze Separate by SCX
Mixture 2
0
200
400
600
800
m/z
Quantitation and protein identification
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