Multidimensional protein identification MudPIT of Urine Proteins

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Overall Goal

• Establish the use of MudPIT for large scale
  protein identification
• Identify novel proteins in urine.

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What is MudPIT?

• A way to simplify the peptide mixture presented
  to the Mass Spectrometer.
• Separates peptides via two dimensional liquid
  chromatography.

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How does MudPIT work?
NNNNNNK
Trypsinized peptide mixture
NNNNNNNNNNNNR
Cation exchange (elution with salt)
HCQMKSTRK
proteome database
Reverse-phase (elution with acetonitrile)
Thousands of mass spectra
ESI/MS-MS
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Platform Validation

• 6 protein mixture standardization
• Human serum albumin (54.31)
• IgG (16.61)
• Antitrypsin (3.38)
• IgA (3.45)
• Transferrin (3.32)
• Haptoglobin (2.94)

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Platform Validation

• 6 protein mixture standardization
• 3 runs of standards identified 787 proteins

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Platform Validation

• 6 protein mixture standardization
• 3 runs of standards identified 787 proteins
• What went wrong?

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Platform Validation

• 6 protein mixture standardization
• Identified 787 proteins on the first run
• Optimization
• Two unique peptides per protein
• Less than 2 potential modification sites per
  peptide
• Delta Cn cutoff gt .1
• Xcorr cutoff

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Platform Validation
Only 3 proteins found that shouldnt exist in
sample.
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Experiment
Load Sample into MudPIT
-Centrifuged
-Concentrated
-Desalted
-Denatured
-Trypsinized
Cation Exchange
Reverse Phase
MS/MS
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Experiment
Hydrophilic peptides
Cation Exchange
Hydrophobic peptides
Reverse Phase
MS/MS
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Experiment
Acetonitrile
Cation Exchange
Reverse Phase
Removes peptides bound to Reverse Phase
MS/MS
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Experiment
Cation Exchange
Reverse Phase
MS/MS
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Experiment
10 Salt Solution
Removes peptides weakly bound to Cation Exchange
resin
Cation Exchange
Reverse Phase
MS/MS
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Experiment
Cation Exchange
Reverse Phase
Not enough hydrophobic residues to bind Reverse
Phase
MS/MS
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Experiment
Acetonitrile
Cation Exchange
Reverse Phase
MS/MS
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Experiment
Cation Exchange
Reverse Phase
MS/MS
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Experiment
Repeat with sequentially higher salt
concentrations followed by Acetonitrile.
Experiment repeated 10 times with 10 urine samples
Cation Exchange
Reverse Phase
MS/MS
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Identified 87 proteins
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Identified 87 proteins
What information should have been included in
this table that wasnt?
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Is this reproducible?
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Is this reproducible?
Acetonitrile
Reverse Phase
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Is this reproducible?
Acetonitrile
Reverse Phase
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Is this reproducible?
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MudPIT vs 2D Gels

• Advantages MudPIT

• Advantages of 2D Gels

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Take Home Messages

• Interpretation of data can have profound effects
  on Mass Spec results.
• In theory MudPIT allows for detection of low
  abundance proteins and is more reproducible than
  2D gels.