Proteomics I 20'12'2002

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Proteomics I - 20.12.2002
Identical Genome but different looks....?
Genome static composition
Proteome highly flexible composition
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Proteomics I - 20.12.2002
What are Proteomics?
Genome
Study of the Proteome
Cell-specific expression
Metabolic state
protein complement of the genome in a cell,
tissue or organism
drugs
Proteome
stress
Culture conditions
proteins can be alternatively spliced and/or
post-translationally modified (PTMs)
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Proteomics I - 20.12.2002
Proteome Analysis
define question to study sample
preparation separation of proteins
identification of proteins quantification of
proteins data base analysis/bioinformatics
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Proteomics I - 20.12.2002
Proteome Analysis
protein degradation protein denaturation changes
of PTM patterns
sample preparation separation of
proteins identification of proteins
quantification of proteins
1D-/2D gel electrophoresis chromatography pull-dow
n experiments
Edman Degradation Mass Spectrometry (MALDI, PSD)
Imaging methods, densitometry ICAT
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Proteomics I - 20.12.2002
DNA (4-letter code) translated into Protein
(21-letter code)
anticodon
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Proteomics I - 20.12.2002
Classification of Amino Acids
I unpolar, aliphatic A, V, L, I
II aromatic F, Y, W
III polar S, T, C, M, N, Q
IV positive charge K, R, H
V negative charge D, E
and there are G and P....
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Proteomics I - 20.12.2002
1. According to Size SDS-PAGE
Separation of Protein Mixtures
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Proteomics I - 20.12.2002
-
SDS
PAGE
PolyAcrylamide Gel Electrophoresis
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Proteomics I - 20.12.2002
2. According to Isoelectric Point
Separation of Protein Mixtures
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Proteomics I - 20.12.2002

• 2D-PAGE
• Isoelectric focusing
• SDS-PAGE

MW
pH gradient
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Proteomics I - 20.12.2002
Advantage 2D-Gel
Disadvantage 2D-Gel
Many proteins are visualized in
parallel Compatible with mass spectrometry Isofo
rms of proteins are separated
Tip of an Iceberg Only high copy number
proteinscan be seen Does not work for very
small proteins Hydrophobic proteins
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What is Mass Spectrometry?
Identification of unknown Proteins

• Separation of molecules according to
• mass to charge ratio (m/z).

Mass Analyzer
Ion Source
Detector
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MALDI
Matrix-Assisted Laser Desorption/ Ionization Mass
Spectrometry

• Sample cocrystallized with acidic matrix
• Mixed ion cloud evaporates into electric field
  (20-30 kV)
• Soft ionization method no fragmentation induced
• Ion competition/Peak compression
• Detection range pmol - amol

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MALDI - TOF Mass Spectrometry
UV Laser
drift area
Detector
Sample
Vacuum 10-7 10-9 Torr
Target with samples
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Proteomics I - 20.12.2002
Detection of Ions Time-of-Flight (TOF) Principle
all ions with identical charge have identical
energy ! kinetic energy is constant between
start and detection
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Proteomics I - 20.12.2002
MALDI samples prepared by different methods
Matrix CHCA
Matrix DHB
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Proteomics I - 20.12.2002
MALDI Sensitivity amol level
Zip Tip
dried droplet
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Purified GST Fusion Protein
kDa
FT
W
E

• Cut out protein band with scalpel
• Washing/shrinking
• Trypsin digest o/n
• Load sample on MALDI Target
• Run MassSpec

116.0

51.8
34.7
30.0
22.0
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A Proteins Fingerprint....

• MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGKWRNKKFELGLE
  FPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAE....

Trypsin, 15 h, 37C, pH 8.0
MSPILGYWK
MH 1094.57/1110.35
MSPILGYWKIK
MH 1335.76/1351.74
GLVQPTR
MH 770.9
MH 2229.11
FELGLEFPNLPYYIDGDVK
HNMLGGCPKERAE....
MH 2739.37
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Fingerprint of Tryptic Digest
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MALDI-Signal

• Isotope Distribution
• 1.1 13C
• Average Mass M
• Monoisotopic Mass MH

m/z
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Calibration
H
external insulin B and angiotensin
internal tryptic peptides (MH 2211.1 and
842.5)
Na
K
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Fingerprint of Tryptic Digest

• write peak list
• remove
• tryptic peptides
• keratin derived peptides
• -data base search
• NCBI nr data base

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Data Base Search
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Results ProFoundSearch
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Rank 1Protein 1
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Rank 1Protein 2
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Fingerprint of GST/M108
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Proteomics I - 20.12.2002
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MS-Fit Data Base Search
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ExPASy
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Proteomics I - 20.12.2002
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Proteomics I - 20.12.2002
What, if there is no protein hit in the data base?

• proteins from non-sequenced organisms
• novel proteins

Possible Solution MS/MS experiments collision
induced fragmentation of a peptide Fragment
analysis gives sequence information
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Post Source Decay
x/y ions
a/b ions
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PSD Spectrum (MALDI)
MH
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Data Base search KIAA 1620
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Proteomics I - 20.12.2002
MALDI Mass Spectrometry and Quantification?
ICAT Isotope Coded Affinity Tag
Principle same chemistry but different mass!
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ICAT Isotope Coded Affinity Tag
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Proteomics I - 20.12.2002
Colorectal Cancer Cell Line (HT-29)
Monolayer
Spheroid culture
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Proteomics I - 20.12.2002
Spheroid culture
Monolayer
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Proteomics II - 10.01.2003
Proteome Analysis II
identification of novel protein-protein
interactions protein networks
quantification of proteins
yeast-two-hybrid pull-down experiments
Systems Biology
Protein Chip Technology