announcements: proposal resubmissions are due 4/23. it is recommended that students set up a meeting...
TRANSCRIPT
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Announcements:• Proposal resubmissions are due 4/23. It
is recommended that students set up a meeting to discuss modifications for the final step of the assignment.
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The Proteome
Using high-throughput methods to identify proteins and to
understand their function
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What is proteomics?
• An organism’s proteome– A catalog of all proteins
• Expressed throughout life• Expressed under all conditions
• The goals of proteomics– To catalog all proteins– To understand their functions– To understand how they interact with each
other
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Protein-protein interactions“The Interactome”
• Yeast two-hybrid analysis• Other “protein complementation” methods• Biochemical purification/Mass
spectrometry
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Purification of interacting proteins
• Immunoprecipitation– Impractical on large scale (identification of
unknowns)• Affinity purification
– Biochemically practical, but too dirty• Tandem affinity purification
– Sufficient yield & purity for identification of unknown proteins
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TAP Purification
Strategy
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Identification of Interacting Proteins
ProteolyticDigestion(Trypsin)
MassSpectrometricAnalysis
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Identifying proteins with mass spectrometry
• Preparation of protein sample– Extraction from a gel– Digestion by proteases — e.g., trypsin
• Mass spectrometer measures mass-charge ratio of peptide fragments
• Identified peptides are compared with database– Software used to generate theoretical peptide mass
fingerprint (PMF) for all proteins in database– Match of experimental readout to database PMF
allows researchers to identify the protein
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Mass spectrometry
• Measures mass-to-charge ratio
• Components of mass spectrometer– Ion source– Mass analyzer– Ion detector– Data acquisition unit A mass spectrometer
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Principle of mass spectrometry
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Ion sources used for proteomics
• Proteomics requires specialized ion sources
• Electrospray Ionization (ESI)– With capillary
electrophoresis and liquid chromatography
• Matrix-assisted laser desorption/ionization (MALDI)– Extracts ions from
sample surface
ESI
MALDI
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Mass analyzers used for proteomics
• Ion trap– Captures ions on the basis
of mass-to-charge ratio– Often used with ESI
• Time of flight (TOF)– Time for accelerated ion to
reach detector indicates mass-to-charge ratio
– Frequently used with MALDI
• Also other possibilities
Ion Trap
Time of Flight
Detector
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A mass spectrum
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Identifying proteins with mass spectrometry
• Preparation of protein sample– Extraction from a gel– Digestion by proteases — e.g., trypsin
• Mass spectrometer measures mass-charge ratio of peptide fragments
• Identified peptides are compared with database– Software used to generate theoretical peptide mass
fingerprint (PMF) for all proteins in database– Match of experimental readout to database PMF
allows researchers to identify the protein
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Limitations of mass spectrometry
• Not very good at identifying minute quantities of protein
• Trouble dealing with phosphorylated proteins
• Doesn’t provide concentrations of proteins• Improved software eliminating human
analysis is necessary for high-throughput projects
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Comparison of Fly Interactome Studies
Yeast Two Hybrid coAffinity Purification/Mass Spec
Pub’d 2003 Pub’d 2011
Done in Yeast Done in S2 cells (embryo hemocytes)
10,623 clones examined 3,488 clones examined
20,405 total interactions 209,912 total interactions
7,048 proteins involved in total 4,927 proteins involved in total
4,780 high confidence interactions 10,969 high confidence interactions*
4,579 proteins in hci 2,297 proteins in hci
586 proteins w/ no previous function
*used different model for confidence
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Summary I
• Goals of proteomics – Identify and ascribe function to proteins under
all biologically plausible conditions• Some accomplishments of proteomics
– For yeast and flies• Yeast two-hybrid method reveals interactome• Subcellular localization of proteins
Long way yet to accomplish overall goals