Proteomics I'

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Proteomics I.

• Methods for High-throughput Protein Separation,
  Identification and Analysis

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Things to know about Proteomics

• Systematic study of Proteomes (the entire
  proteinaceous component of living systems) in a
  single (ideally) experiment
• High-throughput discipline
• Quantitative and exact science
• Very diverse methodology (difficult to master)
• Extensive use of computation

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What is Proteomics good for?

• Profiling of cells, tissues, whole organisms
• Elucidation of mechanisms
• Biomarker discovery and validation
• Drug target discovery and validation
• An experimental platform of choice for Systems
  Biology research

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Separation of Proteins by electrophoresis

• SDS-PAGE or Native PAGE

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Size based separation SDS PAGE

• Coating with SDS assist for solubilization and
  makes all proteins to have the same charge per
  unit length
• The gel has a sieving effect. Smaller proteins
  advance faster.
• The proteins are denatured. The enzymatic
  activities are lost, but the resolution is
  increased because no aggregation occurs between
  the proteins in the sample

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SDS PAGE with Coomassie staining
SDS PAGE with Zn negative staining
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Charge based separation Isoelectric focusing
(IEF)

• Gels are not restrictive
• A pH gradient is established in the gel prior to
  protein separation
• Soluble ampholyte based gradient dynamic and
  transient
• Immobiline based gradient fixed, allows
  focusing to proceed until equilibrium
• The proteins may or may not be denatured.

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pH gradient formation in ampholyte-based IEF
Base
Base
pH 9 8 7 6 5 4
-

Acid
Acid
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pH gradient formation in immobiline-based IEF
Low pI monomer
High pI monomer
Mixer
pH 9 8 7 6 5 4
polymerization
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2D Electrophoresis
First dimension IEF
Second dimension SDS PAGE
pH 10
pH 3
pH 10
kDa 200 100 60 30 10
pH 3
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2D separation of yeast whole cell extract
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imaging
Image Analysis Spot detection
Phosphorimager
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Proteomics II

• Protein identification and analysis by mass
  spectrometry

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Why mass spectrometry?

• Fast
• Sensitive
• Theory of particle electrodynamics is highly
  developed
• Interfaces directly with separation techniques
• Delivers direct identification
• Capable of providing structural information

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Matrix Assisted Laser Desorption Ionizations
(MALDI) MS

• Ionization

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• Detection

E (mv2)/2 (1)
v (2E/m)1/2 (2)
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Protein identification by MALDI MS

• Separate the proteins by 2-DE
• Cut the spots of interest
• Digest with trypsin
• Extract the peptides
• Determine the masses of the peptide fragments
• Search in database

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To determine the masses of the fragments we need
to know the charges. These are determined by
looking at the isotope envelopes of the
corresponding peaks. Adjacent isotope peaks are 1
Da apart in singly charged ions, 0.5 Da apart in
doubly charged etc.
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Identification by peptide fingerprinting

• Since trypsin cleaves only after K and R each
  protein would have unique spectrum of fragments
• Since the genome is sequenced we can deduce the
  theoretical fragment spectrum for all proteins
  encoded in the genome and assemble a database.
  Then we can match the spectrum of our unknown to
  this database
• Since we have computers we can do all of the
  above very fast

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Identification by MS/MS
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Identification of 6xHis-tagged GFP by
nano-LC/MS/MS
MS/MS fragmentation spectrum of peptide FEGDTLVNR
from GFP. The y and b ions are indicated above
corresponding peaks. Mascot peptide score is 53.
A
B
Ni-NTA eluate
500 mM NaCl
100 mM NaCl
200 mM NaCl
50 mM NaCl
Lysate
Wash
Mr (kDa)
FEGDTLVNR
160 -
120 -
100 -
70 -
50 -
35 -
25 -
MS/MS fragmentation spectrum of peptide
SAMPEGYVQER. Mascot score is 51.
A. Bacterially expressed 6xHis-tagged GFP was
enriched by a combination of Ni-NTA affinity
chromatography and anion-exchange chromatography.
Protein fractions were resolved on SDS-PAGE and
the protein bands visualized by negative
imidazol/ZnSO4 staining. The band indicated by
the arrow was cut out of the gel and digested
with trypsin. Tryptic peptides were analyzed by
nano-scale LC/MS/MS using a high-capacity
ion-trap mass spectrometer (Bruker HCT). MS/MS
spectra were matched to protein sequences from
Swissprot using in-house instalation of Mascot
Server (Matrix Science, UK). GFP was identified
with high-score peptides, two of which are shown
on panel B.
SAMPEGYVQER