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.

The Proteome

Using high-throughput methods to identify proteins and to

understand their function

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

Protein-protein interactions“The Interactome”

• Yeast two-hybrid analysis• Other “protein complementation” methods• Biochemical purification/Mass

spectrometry

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

TAP Purification

Strategy

Identification of Interacting Proteins

ProteolyticDigestion(Trypsin)

MassSpectrometricAnalysis

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

Mass spectrometry

• Measures mass-to-charge ratio

• Components of mass spectrometer– Ion source– Mass analyzer– Ion detector– Data acquisition unit A mass spectrometer

Principle of mass spectrometry

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

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

A mass spectrum

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

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

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

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