How we analyzed proteomic data

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How we analyzed proteomic data?

• Session III

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Topics for session III

• Image analysis (for 2-DE gel)
• Mass data analysis
• Protein structure analysis

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1. Image analysis
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Examples of 2-DE results
Healthy control
Patient
MS analysis
Digest to peptide fragment
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Unexpected variation between gels

• Interior variation between 2-DE experiments
• Same loading amount?
• Same gel condition?
• Same staining condition?
• Exterior variation after gel developed
• Unwanted spots (dye or reagent deposit)
• Dirty spots (hair, dust)

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What image analysis software do?

• spot detection
• unwanted spot filtering
• background subtraction
• normalization
• image matching
• expression comparison
• pI/MW calibration
• data organization

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Currently available 2-DE image analysis software

• Melanie 4 (Swiss Institute of Bioinformatics,
  SIB)
• Phoretix 2D (Nonlinear dynamics)
• Progenesis (Nonlinear dynamics)
• Z3 and Z4000 (Compugen)
• Delta2D (Sunergia group)
• A-GelFox 2D (Alpha innotech)
• Flicker (NCI, through internet)

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Spot detection

• One of the first and most important steps in 2-DE
  analysis.
• Locating the spots in the gel image
• defining their shape
• calculating measurement information (volume and
  area)

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Spot detection
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Filtering

• Removal of unwanted spots
• Whats unwanted spots
• dust on gel
• stain deposit
• bulky spots

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Background subtraction

• General background subtraction method
• No background
• Mode of non-spot
• Manual background
• Lowest boundary
• Average boundary

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Normalization

• General normalization method
• Total spot volume
• Single spot
• Total volume ratio

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Image matching
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Expression comparison
Two fold up or down expression are thought to be
significant.
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pI/MW calibration
observed or experimental pI/MW
PI calibration
MW calibration
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Data organization
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Spot annotation
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2. Mass data analysis
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Useful proteomic resource
http//tw.expasy.org/
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Useful proteomic resource
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Matrix Science - Mascot
http//www.matrixscience.com/
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Three major functions in Mascot

• Peptide Mass Fingerprint (PMF) The experimental
  data are a list of peptide mass values from an
  enzymatic digest of a protein. (MALDI-TOF)
• Sequence Query One or more peptide mass values
  associated with information such as partial or
  ambiguous sequence strings, amino acid
  composition information, MS/MS fragment ion
  masses, etc. A super-set of a sequence tag query.
  
• MS/MS Ion Search Identification based on raw
  MS/MS data from one or more peptides. (LC/MS/MS)

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Difference between MALDI-TOF and LC/MS/MS
MALDI-TOF
LC/MS/MS
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2-1 PMF analysis
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Raw data for PMF
m/z
Relative intensity
899.2076 2980.8123 905.2126 1471.3723 909.1917 231
2.2317 915.2181 1533.8486 925.4637 1881.7635 938.3
972 1528.9462 1044.3007 2111.9482 1050.3141 2396.1
550 1060.2797 4689.0698 1066.2889 7302.0029 1072.2
991 5688.8511 1078.3169 8919.1113 1084.2657 1474.5
900 1088.2793 3180.5122 1094.2947 4573.5195 1104.2
638 1546.4652 1110.2837 1470.9734 1271.3163 1498.0
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Mascot PMF query form
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Mascot PMF parameters

• Your name Email
• Search title
• Database
• Taxonomy
• Enzyme
• Monoisotopic or Average
• Modifications
• Protein Mass
• Peptide tol.
• Mass values
• Missed cleavages
• Data file
• Query

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Database
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Taxonomy

• Ensure the hit list will only contain entries
  from the selected species
• speed up a search
• bring a weak match

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Enzyme
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Enzyme

• "None" is not an allowed choice for a Peptide
  Mass Fingerprint, where the specificity of an
  enzyme is essential.
• If the search fails to produce a positive match,
  then try again with semiTrypsin (below) before
  resorting to "None".
• "semiTrypsin" means that Mascot will search for
  peptides that show tryptic specificity (KR not P)
  at one terminus, but where the other terminus may
  be a non-tryptic cleavage. This is a half-way
  house between choosing "Trypsin" and "None".

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Monoisotopic or Average

• nominal mass values calculated from integer
  atomic weights. (H1, C12, N14, O16), not
  practical in proteomics.
• Average mass values equivalent to taking the
  centroid of the complete isotopic envelope
• Monoisotopic mass value the mass of the first
  peak of the isotope distribution.

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Monoisotopic or Average
For peptides and proteins, the difference between
an average and a monoisotopic weight is
approximately 0.06.
Insulin (5.8 kD)
Albumin (66.4 kD)
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Monoisotopic
Tol 1 Da
Monoisotopic MW
-1.01
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Average
Tol 1 Da
Average MW
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Monoisotopic
Tol 2 Da
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Average
Tol 2 Da
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Modifications

• Most protein samples exhibit some degree of
  modification.
• Natural post-translational modifications
  phosphorylation and glycosylation.
• Deliberate modifications deliberately
  introduced during sample work-up, such as
  cysteine derivatisation.

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Modifications
Fixed modifications are applied universally, to
every instance of the specified residue(s) or
terminus. Example Carboxymethyl (Cys) means
that all calculations will use 161 Da as the mass
of cysteine. Variable modifications are those
which may or may not be present. Example if
Oxidation (Met) is selected, and a peptide
contains 3 methionines, Mascot will test for a
match with the experimental data for that peptide
containing 0, 1, 2, or 3 oxidised methionine
residues.
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Modifications
Fixed modifications are applied universally, to
every instance of the specified residue(s) or
terminus. Example Carboxymethyl (Cys) means
that all calculations will use 161 Da as the mass
of cysteine. Variable modifications are those
which may or may not be present. Example if
Oxidation (Met) is selected, and a peptide
contains 3 methionines, Mascot will test for a
match with the experimental data for that peptide
containing 0, 1, 2, or 3 oxidised methionine
residues.
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Peptide tol.
The error window on experimental peptide mass
values
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Missed cleavages
Missed cleavage 0, complete digestion Missed
cleavage gt1, incomplete digestion
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Submit and processing
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Concise protein summary
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protein summary
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PMF protein view (I)
Protein name
Score and Expect
MW and pI
coverage
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PMF protein view (II)
Match peptides
RMS error
No match peptides
Protein information
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2-2 MS/MS analysis
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Raw data for MS/MS
Parent ion
Daughter ion
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Mascot MS/MS query form
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Protein summary
Most possible candidate
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MS/MS Protein view (I)
The sum of all highest scores within each peptide
group
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MS/MS Protein view (II)
Protein score The sum of all highest scores
within each peptide group
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Peptide view
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3. Protein structure analysis
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Research Collaboratory for Structural
Bioinformatics (RCSB)
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Protein data bank (PDB)
Proteosome
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Example 3D structure for proteosome
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Example 3D structure for proteosome
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Example 3D structure for proteosome
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Example 3D structure for proteosome
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Stereo view
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Rotation
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Secondary Structure