Proteomics I

1
Proteomics I

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

2
Proteomics IIfor Monday

• Yeast Two Hybrid
• please study
• Toward a Protein-Protein Map of the Budding Yeast
• (Ito et al., 2000)
• PNAS 97(3), 1143-1147

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Mass Spectrometry

• Molecules to be analyzed, referred to as analytes
  are first ionized (usually in a vacuum),
• Newly charged 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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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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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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MALDI-MS

• ...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
E 1/2 mv2
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Ionization is Variable
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Cipherin
(m/z) mass/charge ratio
M mass of peptide
n2 number of charges
X mass of protons
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Two Formulas, Two Unknowns
...solve for n2,
...then solve for M,
M n2(m/z)2 - X
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Multiple Computations
Each protein yields multiple peptides, with
highly resolvable masses.
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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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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
  inconclussive 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 entire peptide is determined,
• a short amino acid sequence from the peptide.

Often provides enough information to
unambiguously identify the entire protein in
protein, or translated genomic databases.
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b-type ions (a-carboxyl)
y-type ions (a-amino)
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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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...optional reading, available online...
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Isolate Spliceosome Complex Anti-m3G Antibody
Chromotography
Rather than co-precipitating with an anti-m3G
antibody and separating on a gel, use the
anti-m3G antibody to purify the complex using
chromotography.
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Histidine Tag Snp1Nickel Nitrilotriacetic Acid
(Ni-NTA) Affinity Chromotography

• ...Snp1 has been identified as a U1 specific
  snRNP,
• ...the functional Snp1, in the experiment
  (yeast), is carried on a plasmid,
• ...it has been modified by a hexahistidine
  addition to the N-terminus,
• ...the exposed his-tag binds to lead on a
  chromatography column, is washed off with
  imidazole.

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Separate Ni-NTA Eluate

• ... SDS-PAGE fractionation yields 20 proteins,
• bands are in-gel digested,
• extracted,
• send to MS-MS for ID.

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Identify U1 Specific Bands
Glycerol Centrifugation to Separate after Ni-NTA.
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MS-MS

• 1. fragment at 756.9 was selected for sequencing,
• 2. LEEL sequenced,
• 3. AELEELEEFEFK unique match in the database.

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Whole Complex De-convolution
Prp39p (106 aa peptide)

• Prp40p (41 aa peptide)

• ...peptides can be isolated and from heterogenous
  samples, sequenced and IDd.
• Prp39p and Prp40p

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Found Players

• All of the known U1-specific proteins found to
  date were identified,
• An ortholog to human U1-C was found that had not
  been identified in yeast was detected,
• Four novel proteins were also identified.

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Establishment of Principle

• ...so, in one fell swoop, years of molecular
  genetic and biochemical research was replicated,
• and
• ...a U1-C protein, not detected above, was
  identified as a component in the system,
• and
• ...four novel proteins have been IDd,
• not necessarily components,
• but excellent research leads.

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Use of Principle

• ...in one subsequent experiment with isolated
  human spliceosome,
• 70 spliceosome protein spots were analyzed,
• 19 novel splicing factors were identified,
• and
• ...all 19 could be cloned directly via EST
  libraries,
• and
• ...in vivo conformation of the role of these
  factors in splicing was obtained through
  co-localization studies using green fluorescence
  protein (GFP).

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Quantitation

• ...progress has been made in using MS to quantify
  protein expression,
• requires labeling of one or more protein species
  and is generally limited to relative expression.

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Signaling Pathways(4.2)
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Organelle(4.3)
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please study Functional Genomics by Mass
Spectrometry (Andersen and Mann, 2000) FEBS
Letters 480, 25-31
Neubauer et al. available online or reference.