Transcript Applying Proteomics in Biomedical Research

Using Proteomics for
Biomedical Research
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.”
Examples of Proteomes:
ribosomes
yeast
nucleosomes
transcriptional complexes
biopsy samples
affinity – chemical or biological interactions – IP etc.
2-D gel
rat brain homogenate
1 mg protein load
Resolved over 2000 spots
pI and MW of protein spots
2-D gel of an
uncharacterized bacteria
collected from
Yellowstone National Park
Mammoth Hotsprings
October 2007
(Collaboration with Bruce
Fouke, UIUC Geology)
Interpreting 2-D gel images
•Image warping
•Normalization
•Statistical significance
•Real spots versus artifacts
•Internal standards
•Gel to gel variations
•Sensitivity
“You’ve got one protein missing …”
“No, you’ve one extra protein !”
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
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)
Protein
abundance
in cell
varies
by 5-6
orders !!
Bruker Daltonics
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)
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
Fractionation (Agilent method)
Agilent
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
Functional Specific Protein Stains
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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
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
Cy3 channel
Cy5 Channel
Channel Cy3 and Cy5 superimposed
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
MALDI ToF MS
Matrix Assisted Laser Desorption Ionization Time-offlight mass spectrometer
ESI MS
Electro-Spray-Ionization mass spectrtometer
Schematic of a modern MALDI-ToF MS using DHB as matrix
Matrix for MALDI ToF
• 2,5-dihydroxybenzoic acid (DHB)
• α-cyano-4-hydroxy-cinnamic acid
• 3,5-dimethoxy-4-hydroxycinnamic acid
(sinapinic acid)
• Specific compounds for glycoprotein etc
Schematic of ESI MS developed by John Fenn (taken from
Fenn’s Nobel Prize lecture)
ETD (Electron Transfer Dissociation)
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anion reagent – Fluoranthene
fragmentation is superior over CID
essential for difficult peptides with PTM
enables structural analysis of complex
carbohydrate
Dual source ESI for LTQ-Orbitrap
Thermo LTQ
CID
ETD
Waters Q-ToF MS
Waters Q-ToF (Quadrupole time-of-flight mass
spectrometer)
Quantitative Proteomics
• Relative Quantitation
• Absolute Quantitation
• Typically the answer is the presence or
absence of a certain protein
• Expression
• Interaction
• Modifications
Top Down Proteomics
• 2-D Gel electrophoresis (DIGE)
• Mass spec based top down
measurement (including using
traditional fractionation methods)
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 !
Bottom Up Proteomics
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Label Free mass spec method
MudPIT
Waters High – Low method
Stable-isotope labeling method
Metabolic
ICAT
iTRAQ
O-18
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)
ICAT
Applied Biosystems Q-Trap 5500
•hybrid triple quad/ion-trap MS
•6 orders of dynamic range
•Resolution 3000
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 proteinprotein 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
Specific Proteomics Reactor
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Daniel Figeys at Ottawa, Canada
Glycomics Reactor
Phosphorylation Reactor
Ubiquitin Reactor
Interactome Reactor
Thermo LTQ-FT-ICR-MS
•attomole
sensitivity
•widest dynamic
range ( > 4,000)
•ppb mass
accuracy
•resolution
>750,000
•ECD
Top Down Proteomics
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McLafferty (Cornell)
Neil Kelleher (formerly UIUC Chemistry)
Uses FT-ICR-MS
Middle down approach (mild digestion)
Pro-cite program
Thermo-LTQ-Hybrid/Orbitrap
Thermo-Orbitrap
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
Using Ion Mobility MS to differentiate neuropeptides
differing by one D/L amino acid
• YdAEFL amide
• YlAWFL amide
• FdMRF amide
• FlMRF amide
Comparison study between
• Thermo LTQ,
• Thermo LTQ-Orbitrap
• Waters SYNAPT
(Slide taken from Bruker Daltonics)
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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Images obtained from a
mouse kidney section.
(a) digital photograph
(b) total ion count
(c) Ion 4,965 Da
(d) Ion 11,362 Da
MALDI Imaging (taken from Waters literature)
Proteomics - Challenges
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Sample
Sampling Handling
Complexity Reduction
Chromatography
Mass Spectrometry
Bioinformatics
(Abersold (2009) Nature Methods, vol 6, 411)
Proteomics Center
Carver Biotechnology Center
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 [email protected])