Using Proteomics for Biomedical Research

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Using Proteomics for Biomedical Research
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Proteomics coined by Wilkins and Williams .
The study of proteins as an result from
genomics Ruedi Abersold defining the
mandate of proteomics proteomics represents
the effort to establish the identities,
quantities, structures and biochemical and
cellular functions of all proteins in an
organism, organ, or organelle, and how these
properties vary in space, time and physiological
state.
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Examples of Proteomes ribosomes
yeast nucleosomes transcriptional complexes
biopsy samples affinity chemical or biological
interactions IP etc.
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2-D gel rat brain homogenate 1 mg protein
load Resolved over 2000 spots pI and MW of
protein spots
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2-D gel of an uncharacterized bacteria collected
from Yellowstone National Park Mammoth
Hotsprings October 2007 (Collaboration with
Bruce Fouke, UIUC Geology)
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• Interpreting 2-D gel images
• Image warping
• Normalization
• Statistical significance
• Real spots versus artifacts
• Internal standards
• Gel to gel variations
• Sensitivity

Youve got one protein missing No, youve
one extra protein !
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• Human Genome
• Nucleic Acids
• 30,000 genes
• DNA is localized in the nucleus, simple
  extraction !
• Proteins extraction is always a challenge
  !
• localization (compartmentalization e.g.
  nucleus)
• solubility, salt, pH

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• More on protein extraction challenges
• Membrane proteins (hydrophobic)
• Glycoproteins
• Timing of protein extraction (e.g. cyclins)
• Active versus inactive forms
• Isozymes
• Post-translational modifications (over 300)

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Protein abundance in cell varies by 5-6 orders !!
Bruker Daltonics
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Fractionation

• Protein fractionation and purification is a key
  step
• in proteomics studies
• Whole animal organ tissue laser captured
  cell clusters.
• Whole cell extract nuclear or cytoplasmic
  extract
• Whole brain hippocampus post-synaptic
  membranes
• Avoid protein degradation (proteolysis
  inhibitors)
• For studies involving PTMs (e.g. phosphorylation
  use
• phosphatase inhibitors)
• Chromatography (ion exchange, HPLC, affinity)
• Avoid inadvertent chemical modifications
  (carbamate,
• formyl or acetyl from acid treatment.. etc)

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• Common techniques for proteomics
• Fractionation and isolation (extraction,
  centrifugation,
• chromatography)
• Classical methods such as Edman Sequecing, amino
  acid analysis
• Electrophoresis
• Immunological methods antibodies pull-down
  (IP),
• ELISA, western blot, immuno-histology,
  bead-based Luminex assays
• Structural methods, scattering, microscopy, NMR
• Gel based versus chomatography versus mass spec
• Spectroscopy (VIS/UV), Mass spectrometry

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Fractionation (Agilent method)
Agilent
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Beckman PF-2D

• First dimension - chromatofocusing
• Chromatofocusing is a column based chromatography
  method separation proteins according to their pI
• (similar to 2-D gel)
• Second dimension is RP-HPLC
• This method is non-gel based

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Functional Specific Protein Stains

• Invitrogen (Molecular Probes)
• Pro-Q Diamond phosphorylation
• Pro-Q-Emerald glycosylation
• Quantitative, 3 orders
• Good sensitivity
• Works well with SYPRO-Ruby
• Compatible with mass spec

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DIGE

• Fluorescence Difference in Gel electrophoresis
• Protein samples labeled with CyDyes
• Cy3 for Sample 1
• Cy5 for Sample 2
• Cy2, normalization, Samples 1 and 2
• All three labeled samples pooled and run in the
  same gel

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Cy3 channel
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Cy5 Channel
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Channel Cy3 and Cy5 superimposed
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Mass spectrometry to Proteomics is like PCR to
Genomics
2002 Nobel Prizes in Chemistry Mass spectrometry
for macromolecules "for their development of soft
desorption ionisation methods for mass
spectrometric analyses of biological
macromolecules"
Koichi Tanaka
John B. Fenn
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MALDI ToF MS Matrix Assisted Laser Desorption
Ionization Time-of-flight mass spectrometer ESI
MS Electro-Spray-Ionization mass spectrtometer
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Schematic of a modern MALDI-ToF MS using DHB as
matrix
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Matrix for MALDI ToF

• 2,5-dihydroxybenzoic acid (DHB)
• a-cyano-4-hydroxy-cinnamic acid
• 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic
  acid)
• Specific compounds for glycoprotein etc

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Schematic of ESI MS developed by John Fenn (taken
from Fenns Nobel Prize lecture)
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(No Transcript)
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ETD (Electron Transfer Dissociation)

• anion reagent Fluoranthene
• fragmentation is superior over CID
• essential for difficult peptides with PTM
• enables structural analysis of complex
• carbohydrate

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Dual source ESI for LTQ-Orbitrap
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Thermo LTQ
CID ETD
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Waters Q-ToF MS
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Waters Q-ToF (Quadrupole time-of-flight mass
spectrometer)
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Quantitative Proteomics

• Relative Quantitation
• Absolute Quantitation
• Typically the answer is the presence or absence
  of a certain protein
• Expression
• Interaction
• Modifications

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Top Down Proteomics

• 2-D Gel electrophoresis (DIGE)
• Mass spec based top down measurement (including
  using traditional fractionation methods)

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Middle Down Proteomics

• Spear-headed by Neil Kelleher
• Uses mild digestion (enzymatic or chemical
  cleavages)
• CNBr cleaves at methionine
• Acetic Acid, Formic Acid - cleaves at aspartic
  acid
• Formylation and acetylation precaution !

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Bottom Up Proteomics

• Label Free mass spec method
• MudPIT
• Waters High Low method
• Stable-isotope labeling method
• Metabolic
• ICAT
• iTRAQ
• O-18

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Absolute Quantitation

• Typically using a Triple-Quadrupole MS
• Add known amount of specific stable isotope
  labeled peptide
• Also known as Accurate Mass Tagging method (R.
  Smith)

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ICAT
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Applied Biosystems Q-Trap 5500

• hybrid triple quad/ion-trap MS
• 6 orders of dynamic range
• Resolution 3000

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Interactome

• Cell Research Vol 18 No 7 July 2008
• Mapping the human protein interactome
• Daniel Figeys - The Ottawa Institute of Systems
  Biology, The Department of Biochemistry,
  Microbiology and Immunology, University of
  Ottawa,
• Ottawa, ON, K1H 8M5, Canada
• Interactions are the essence of all biomolecules
  because they cannot fulfill their roles without
  interacting with other
• molecules. Hence, mapping the interactions of
  biomolecules can be useful for understanding
  their roles and functions.
• Furthermore, the development of molecular based
  systems biology requires an understanding of the
  biomolecular
• interactions. In recent years, the mapping of
  protein-protein interactions in different species
  has been reported, but
• few reports have focused on the large-scale
  mapping of protein-protein interactions in human.
  Here, we review the
• developments in protein interaction mapping and
  we discuss issues and strategies for the mapping
  of the human protein

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Specific Proteomics Reactor

• Daniel Figeys at Ottawa, Canada
• Glycomics Reactor
• Phosphorylation Reactor
• Ubiquitin Reactor
• Interactome Reactor

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Thermo LTQ-FT-ICR-MS

• attomole
• sensitivity
• widest dynamic
• range ( gt 4,000)
• ppb mass
• accuracy
• resolution
• gt750,000
• ECD

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Top Down Proteomics

• McLafferty (Cornell)
• Neil Kelleher (formerly UIUC Chemistry)
• Uses FT-ICR-MS
• Middle down approach (mild digestion)
• Pro-cite program

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Thermo-LTQ-Hybrid/Orbitrap
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Thermo-Orbitrap
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Waters Q-ToF SYNAPT G2

• Newest member of Waters high resolution Q-ToF
• family of mass spectrometers
• First commercial Mass Spectrometer capable of
  measuring ion-mobility
• Uses T-Wave (traveling wave) to improve ion
  mobility
• T-Wave uses RF
• Separation by mass, charge and shape

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Using Ion Mobility MS to differentiate
neuropeptides differing by one D/L amino acid

• YdAEFL amide
• YlAWFL amide
• FdMRF amide
• FlMRF amide

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• Comparison study between
• Thermo LTQ,
• Thermo LTQ-Orbitrap
• Waters SYNAPT

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(Slide taken from Bruker Daltonics)
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Jonathan Sweedler Laboratory (UIUC) Micro-sampling
technique

• Sampling of neuropeptides using SPE beads
• Aplysia abdominal bag cell clusters
• PAC (Pleural Abdominal Connective) nerve
• stimulation
• Beads placed before, during PAC stimulation
• Neuropeptides varies many orders, verified by MS

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AA

• Images obtained from a
• mouse kidney section.
• digital photograph
• total ion count
• Ion 4,965 Da
• Ion 11,362 Da

MALDI Imaging (taken from Waters literature)
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Proteomics - Challenges

• Sample
• Sampling Handling
• Complexity Reduction
• Chromatography
• Mass Spectrometry
• Bioinformatics
• (Abersold (2009) Nature Methods, vol 6, 411)

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Proteomics Center Carver Biotechnology Center
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Lab Tours

• Wednesday, September 1, 11 AM 12 Protein
  Sciences Facility (Noyes Laboratory 315 (SW
  Corner)
• Monday, September 6, Happy LABOR DAY!
• Wed, Sept 8, meet in Mumford 11 AM short
  lecture then go to IGB for tour.
• Questions and Inquiries
• Peter Yau (333-3841 pmyau_at_illinois.edu)