Proteomics I

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Proteomics I

• Rob Mitra

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What is Proteomics???

• A fuzzy set of techniques in which a large number
  of proteins are analyzed in one experiment.
• - Protein Abundance Levels
• - Protein Localization
• - Protein-Protein Interactions (complexes,
  partners)
• - Post-Translational Modifications

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Proteomics is a Tough Business!!

• Proteins are found in complex mixtures
• 6-8 orders of magnitude variation in abundance
• No protein equivalent of PCR technique!!!
• No De Novo Design of Binding Partners
• Proteins Have Diverse Characteristics

Genomic Scientist
Proteomic Scientist
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So Why Bother?
DNA
Proteins
Proteins do the work in most cellular processes!
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So Why Bother? Part II
Compare Protein Levels
To mRNA Levels SAGE.
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So Why Bother? Part II cont.
Big Graph Looks Reasonable! But look at
inset. Same mRNA 20 fold variation in protein
levels
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So Why Bother? Part II cont.
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So Why Bother??? Part III
From Mann, Nat Biotech 2003
Post-Translational Modifications! These regulate
a proteins activity,localization,turnover,intera
ctions
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The Proteomics Tool Box

• Mass Spectrometry
• Protein Chips
• Surface Plasmon Resonance
• Peptide/Protein Display

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Mass Spectrometry Lots of Acronyms!

• Multiple Steps to Protein Analysis with Mass
  Spectrometry.
• Each Step Has Many Flavors
• Procedure is Modular

Protein Or Peptide Mixture
Mass/Charge Histogram
MS2
Separation
Ionization
MS
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Keep it Simple

• A Single Flavor LC-ESI Tandem Mass Spectrometry
• Understanding the Output
• Variations on a Theme
• Examples!

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How Does a Mass Spec Work?
Several Complex Mixtures of Peptides
Complex Mixture of Ions
Very Complex Peptide Mixture TRYPSIN!
Mass/Charge Histogram
Separation Reverse Phase Liquid Chromatography
Ionization Electrospray Ionization
Mass analyzer
Tandem Mass analyzer
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Reverse Phase Liquid Chromatography
Mixture of Peptides
Step 1 Bind to Matrix
Hydrophobic Matrix
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Reverse Phase Liquid Chromatography
50 Acetonitrile
Step 2 Elute with Organic Gradient
Hydrophobic Matrix
More Hydrophobic Peptide
More Hydrophilic Peptide
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Reverse Phase Liquid Chromatography
100 Acetonitrile
Step 2 Elute with Organic Gradient
Hydrophobic Matrix
Hydrophobic Peptide
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Each Output Fraction is Less Complex than the
Input
More Hydrophilic
More Hydrophobic
Absorbance
Time (min)
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How Does a Mass Spec Work?
Several Complex Mixtures of Peptides
Complex Mixture of Ions
Very Complex Peptide Mixture
Mass/Charge Histogram
Separation Reverse Phase Liquid Chromatography
Ionization Electrospray Ionization
Mass analyzer
Tandem Mass analyzer
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Electrospray Ionization
From http//www-methods.ch.cam.ac.uk/meth/ms/theo
ry/esi.html
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Ions
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Electrospray Ionization

• Biomolecules in multiple charge states
• Small volumes (10 nanoliter/minute) flow rates
• Mixture of Ions is injected into mass analyzer at
  a high velocity

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How Does a Mass Spec Work?
Several Complex Mixtures of Peptides
Complex Mixture of Ions
Very Complex Peptide Mixture
Mass/Charge Histogram
Separation Reverse Phase Liquid Chromatography
Ionization Electrospray Ionization
Mass analyzer
Tandem Mass analyzer
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Theory Break

• F z(E v x B)
• F ma

Lorentz Law
Newtons Law
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Back to Lorentz Law
Radius of turn depends on two parameters M/Z and
B!
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Magnetic Sector Mass Analyzer
Close cousin of quadrupole mass analyzer (more
common)
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The M/Z Histogram
From Gygi et al. 1999
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Quadrupole analyzer the cousin to magnetic
sector analyzers
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A Map is Like a 2D Peptide Gel
First Dimension Reverse Phase
Chromatography Separation By Hydrophobicity
RT
min
m/z
Courtesy of Kyriacos Leptos
Second Dimension Mass Spectrometry Separation
by Mass
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Bovine Serum Albumin
RT
min
Courtesy of Kyriacos Leptos
m/z
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A 3D View of Peptide DAFLGSFLYEYSR
abundance
rt
m/z
Courtesy of Kyriacos Leptos
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What Information Can Be Extracted From A Single
Peptide Peak
Isotopic Variants of DAFLGSFLYEYSR
abundance
rt
m/z
_at_ 36.418 min
abundance
0 X 13C
1 X 13C
2 X 13C
Courtesy of Kyriacos Leptos
3 X 13C
m/z
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Bovine Serum Albumin
RT
min
Courtesy of Kyriacos Leptos
m/z
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How Does a Mass Spec Work?
Several Complex Mixtures of Peptides
Complex Mixture of Ions
Very Complex Peptide Mixture
Mass/Charge Histogram
Separation Reverse Phase Liquid Chromatography
Ionization Electrospray Ionization
Mass analyzer
Tandem Mass analyzer
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Tandem Mass Spectrometry

• Goal - For each peptide peak in the map,
  determine the peptide sequence using a clever
  idea.

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Tandem Mass Spectrometry
Quadrople Q1 scans or selects m/z. Q2 transmits
those ions through collision gas (Ar). Q3
Analyzes the resulting fragment ions.

• Siuzdak, Gary. The emergence of mass
  spectrometry in biochemical research. Proc.
  Natl. Acad. Sci. 1994, 91, 11290-11297.
• Roepstorff, P. Fohlman, J. Biomed. Mass
  Spectrom. 1994, 11, 601.

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The M/Z Histogram
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Peptide Fragmentation and Ionization
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Collision Induced Ionization of Peptide
B ions
Y ions
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Lets look at B ion spectrum
Abundance
115.11
202.19
259.24
M/Z
Asn
114.11 daltons
87.08 daltons
Asn Ser
Asn Ser Gly
57.05 daltons
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Tandem Mass Spectra Analysis
y b
Gygi et al. Mol. Cell Bio. (1999)
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In Practice, Its Tricky

• Different types of ions (e.g. a and z)
• Post-translational modifications
• Isotopes
• Noise
• Not all fragmented species present
• Ions may lose a water or ammonia molecule

Use parent ion mass, spectra, protein and genomic
databases
Software solutions SEQUEST, others