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Protein Interaction ReportersProtein Interaction ReportersPaper: X.Tang, J.Bruce,A new crosslinking strategy: protein interaction reporter (PIR) technology for protein–protein interaction studies Mol.BioSyst., 2010, 6, 939–947
PPIs
Protein Protein Interactions
Introduction to PPI principles
• PPIs at work in Signaling:• Recruit signaling complex
1. Δ Conformation2. affecting activity / Kd, 3. additional PPI‘s
• Proteins: key to bio-activity• Dynamic cell: Δshape,
Divide, Metabolism• e.g. Neuron vs. Hepatocyte
Δ…
”ch
an
ge”
in
PPI thought-experiment
• Imagine a cell suddenly devoid of PPIs:• No signaling: a cell in “darkness“• No nuclear wall, no cytoskeletton, no RME
(receptor mediated endocytosis), no signalosomes, Histone Mod, PTMs etc...
• The unlucky cell would be rendered deafblindparalytic, and wouldultimately disintegrate
alpha-Keratin
Basics of Binding
• Binding affinity: ~ free energy• Gibbs-Helmholz: • A + B AB
Binding continued
• Δ G = Δ Ecol + Δ Gdes –TΔSsc + ΔGrot/trans
• Hurdles obtaining (good) Data:• Intrinsically unstructured Prot.• Lack of crystallographic data• Different environments• Transient interactions• Time consuming
Importance to study PPIs
• Classic Principle of Science: Observation Hypothesis
Theory Models Data• Now: lots of Data Data Intensive Science• PPI studies yield a who is who type of
understanding (relationships)• Some Fields relying on PPI data:
• Theoretical Biology ←→ Systems Biology
• Evolution- / EvoDevo- science
• Drug Dev: tumor suppressors, DNA repair,...
• Drug Design: Docking, Receptors, targets (e.g. hERG)
How is PPI data obtained?
• numerous methods e.g. Yeast two hybrid• Y2H is high throughput amenable• High Throughput methods kicked off
massive PPI data collection• Databases : BIND, DIP, PPIS, PPI, GRID
(General Repository for Interaction Datasets)
The current status of DIP DB
• #Number of proteins 23200
• #Number of organisms 372
• #Number of interactions 71275
• #Number of distinct experiments: 69470 • #Number of data sources : 4606
Nov 2010
MASS SPECTROMETRY BASED PPI
Protein Interaction Reporters
• Chemical crosslinking (well known)• bona-fide interactions stabilized• Mass spec analysis• Requirements:
• Identification of the protein interaction network• Known Low-res interaction mapping of Protein-
complex
Alternative PPI Analysis
-• more or less labor intensive• introduce system pertubations
• i.e. No System-wide snapshot possible!
+• may be easier in data-evaluation (for now)
Complex sample matrix &MSMS data
• Identification is non-trivial even when purified• Resulting Cross-linked sample species includes:
• Unmodified peptides
• Inter cross linked peptides
• Intra cross linked peptides
• Dead end labeled peptides
• Multiply labeled peptides
• Nonspecific labeled peptides
• Low abundance of cross-linked species
• Data complexity of MS-MS / MSn spectra• Fragment ions of “peptide versions”
• modified peptide version: Partial/full crosslinked peptide-two still attached
• Additional PTMs
Fig. Species and fragment outcome
What is a good PIR?• self sufficient cross-linking:
• Detection of a cross link should contains the information of a PPI interaction and topology: e.g. site X and Y in close proximity?
• PIR vs. Immunoprecipitation methods:• Susceptible to nonspecific interaction with non POIs
(Proteins of Interest), ABs, solid support
• Sensitivity range improved by Affinity methods:• Highly abundant cross-linked peptides extracted with
an affinity-support for such species (e.g. pulldown)
• Structural models can be aided through obtained low-res topologies
Data Complexity• Problem: multiple cross linked species → digested to
n peptides, can lead to → n² cross-linked species
• Knowledge of topology and binding can lead to faster drug-dev Small mol. Drug-antagonist pipeline
• Data complexity reduced by:• Enrichment of cross-linked species
• Introduction of physical and software signature patterns
→ Data mining (not discussed)
• Physical Strategies:• Affinity tagged spacer region
• Isotope labeling
• Chemical cleavable bonds
• Mass spec cleavable bonds
Previous cross-linkers
• Protein fragments, cross link fragmentsprevents PPI analysis• →Spectral complexity
• Good cross linker allow seq. analysis:• 1st Measure cross linked peptide complex• 2nd Dissociate the cross linker yielding:
• two intact peptides + • cross-linker signature
• → sequence peptides and align with the database
Protein Interaction reporters (2005)
• Labile bond in the cross linker is MS cleavable (in situ)
Tunable:•labile bonds•reaction groups•affinity groups
Next step is sequencing
• Each peptide can be sequenced by either• Accurate mass (HR) measurement (e.g. Orbitrap)• By MS-MS fragmentation
• Intact peptide mass allows standard protein identification methods
• Labile bond, cleave by CID:• E.g. Based on aspartic acid and proline bond (D-P)
• By MALDI laser: BIPS-Matrix• Photocleavable: via online-UV irradiation after
elution/separation from the LC system → the separated complex co-elutes
Next step is identification
• Identify: • 1st cross-linked ions • 2nd protein of origin
• 1. Identify • which ions contain a cross link and its • type (inter, intra, dead end labeled)
• Predictable mass relationships reveal type of interaction
• False Discovery Rate (2010): FDR >6%
multiplexed FTICR-MS Spectra
• Inter cross link determination…
Identification/rel. quantitation
• unbiased identification of PPIs of intact cells may be possible:1. Cross link live cells
2. Lyse to extract proteins
3. Digest & preclean: affinity capture of cross linked peptides
4. LC/MSn analysis:• measure and then CID to cleave the complex
(CID…Collision induced dissociation)
PIR isotope label
• PIR isotope-label differs in 6-8Da:• relative quantitation via observed peak
intensities→method similar to ICAT, ITRAQ
Future outlook & Summary• Functionally directed PIRs:
• to label specific classes of proteins and their PPIs
• Reaction mechanism-selective cross-linkers • e.g. Adenosine-analog based cross linker to identify Kinase-
substrate pairs
Summary: