October 10, 2002, Thursday

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• October 10, 2002, Thursday
• NATIONAL DESK
• 3 Whose Work Speeded Drugs Win Nobel
• By KENNETH CHANG (NYT) 1067 words
• Three scientists share this year's Nobel Prize in
  Chemistry for developing techniques to identify
  and map proteins, carbohydrates, DNA and other
  large biological molecules. The techniques have
  sped the development of drugs and could lead to
  quicker diagnosis of cancer. They helped create a
  new field of biology, proteomics, in which
  scientists are trying to catalog the interplay of
  hundreds of thousands of proteins in human cells.
  
• Dr. John B. Fenn, 85, a research professor at
  Virginia Commonwealth University in Richmond, and
  Koichi Tanaka, 43, an engineer at the Shimadzu
  Corporation in Kyoto, Japan, share half of the 1
  million prize. Working independently, they
  improved a technique known as mass spectrometry
  to identify proteins by how quickly they are
  accelerated in an electric field.
• Now, biologists can determine the proteins in a
  sample in seconds rather than weeks. Mr. Tanaka
  is one of the youngest chemistry laureates, while
  Dr. Fenn did not begin his Nobel-winning research
  until he was in his 60's. ''I was dumbstruck,''
  Dr. Fenn said of receiving the phone call from
  the Royal Swedish Academy of Sciences at 530
  yesterday morning. ''I'm still in a daze. I'm not
  entirely sure I'm coherent.''
• Dr. Kurt Wüthrich, 64, a professor of biophysics
  at the Swiss Federal Institute of Technology in
  Zurich, received the other half of the prize for
  using nuclear magnetic resonance -- the same
  underlying science as in the familiar medical
  M.R.I. -- to map the structure of proteins.

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Proteomics I

• Mass Spectrometry
• please study
• Functional Genomics by Mass Spectrometry
• (Andersen and Mann, 2000)
• FEBS Letters 480, 25-31

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Proteomics IIfor Wednesday/Friday

• Yeast Two Hybrid
• please study

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2-D Gel
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Mass Spectrometry

• Molecules to be analyzed, referred to as analytes
  are first ionized (usually in a vacuum),
• Newly charged (protonated) molecules are
  introduced into an electric and/or magnetic field
  in gas phase,
• Their path through the field is a function of the
  mass to charge ratio m/z,
• m/z of the ionized species can be used to deduce
  the mass of the analyte with high precision.

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Ionization is Variable
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Cipherin
(m/z) mass/charge ratio
M mass of peptide
n number of charges
X mass of protons
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Two Formulas, Two Unknowns
...solve for n,
...then solve for M,
M n(m/z)2 - X
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Multiple Computations
Each protein yields multiple peptides, with
highly resolvable masses.
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Biological Samples

• ....bringing polypeptides and nucleic acids to
  the gas phase usually degrades the molecules,

1988
matrix assisted laser desorption/ionization mass
spectrometry MALDI-MS
electrospray ionization mass spectrometry ESI-MS
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MALDI-MSMatrix Assisted Laser Desorption
Ionization

• ...peptides are suspended in a matrix of
  light-absorbing molecules,
• deposited onto a solid substrate,
• high-voltage is applied to the solid substrate,
• laser excitation of the matrix,
• peptides are released from the matrix, and
  accelerate through the electrical field,
• ionized occurs during desorption.

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MALDI-MS
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MALDI Peptide Mass MappingMass Fingerprinting

• ...proteins are cleaved in a sequence specific
  manner,
• thus, each protein in a proteome has a unique
  peptide mass subset,
• these subsets can be computationally derived from
  protein databases, and translated genomic DNA
  sequences,
• experimentally determined unknowns can be
  compared, via computers, to online databases for
  identification,
• ..scalable, multiple samples can be deposited at
  once, computers sort out the constituents.

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Proteases

• ...proteins are first degraded into smaller
  peptides by sequence specific proteases,
• assists in elution from gels and other sources,
• large polypeptides give indefinite masses.

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Figure 1a.
mass
MALDI MS Mass Fingerprinting.
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However

• ...protein databases are not yet inclusive,
• protein fingerprint data is not available, or is
  inconclusive for large parts of most genomes,
• ...some proteins are too small to give enough
  peptide fragments for fingerprinting,
• ...computer deconvolution has its limits.

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Electrospray Ionization Mass SpectrometryESI-MS

• Peptides analytes, in solution, are passed
  through a charged needle that is kept at high
  electrical potential,
• the peptides are ionized,
• this disperses the the solution into a fine
  spray,
• the solvent quickly evaporates,
• peptides now in gas phase,
• Enter mass spectrometer for mass fingerprinting,
• or
• Peptide Sequencing.

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ESI-MS
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Figure 1b.
mass
Mass Spectrometry via Electro-Spray Ionization
(ESI-MS).
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Tandem Mass Spectroscopy(MS-MS)

• ...mass spectrometry can also be used to obtain
  sequence to identify peptides,
• treatment with sequence specific proteases
  provides information of the terminal residues,
• the mass of the peptide fragment is determined,
• a short amino acid sequence from the peptide is
  obtained.

Often provides enough information to
unambiguously identify the entire protein in
protein, or translated genomic databases.
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b-type ions (a-amino)
y-type ions (a-carboxyl)
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Figure 1c.
693.37(EYL)1098.55

total peptide mass

TQLYEYLQR
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MALDI Dual QuadrupoleMALDI MS-MS
Combines MALDI-MS scalability with ESI-MS
sequencing.
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Genome Searching

• ...we now have the ability to match heterologous
  MS data to raw genomic data,
• i.e. unannotated, untranslated DNA sequence from
  the genome projects,
• i.e. dont need complete protein sequences for
  fingerprinting.

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Multi-protein Complexes

• ?

...i.e. nuclear pore complexes, ...i.e. cellulose
synthase complexes, ...i.e. spindle pole
apparati, ...i.e. proteins involved in the
spliceosome, etc.
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Signaling Pathways(4.2)
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Organelles(4.3)