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: