This document discusses protein interaction reporters (PIRs), a crosslinking strategy to study protein-protein interactions (PPIs) using mass spectrometry. PIRs chemically crosslink interacting proteins in their native state, then use a cleavable linker and mass spectrometry to identify and sequence the interacting proteins. Key advantages of PIRs include their ability to provide system-wide snapshots of PPI networks, introduce isotopic labels for relative quantification, and enrich for crosslinked peptides to reduce data complexity challenges. Future directions may include developing PIRs targeted to specific classes of proteins or reaction mechanisms to gain more functional insight into PPIs.

1. Protein Interaction Reporters
Paper: X.Tang, J.Bruce,A new crosslinking strategy:
protein interaction reporter (PIR) technology for
protein–protein interaction studies Mol.
BioSyst., 2010, 6, 939–947

2. PPIs
Protein Protein Interactions

3. Introduction to PPI principles
• PPIs at work in Signaling:
• Recruit signaling complex
Δ…”change”
1. Δ Conformation
2. affecting activity / Kd,
3. additional PPI‘s
in
• Proteins: key to bio-activity
• Dynamic cell: Δshape,
Divide, Metabolism
• e.g. Neuron vs. Hepatocyte

4. 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
deaf alpha-Keratin
blind
paralytic, and would
ultimately disintegrate

5. Basics of Binding
• Binding affinity: ~ free energy
• Gibbs-Helmholz:
• A+B AB

6. 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

7. 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)

8. 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)

9. The current status of DIP DB
• #Number of proteins Nov 2010
23200
• #Number of organisms
372
• #Number of interactions
71275
• #Number of distinct experiments:
69470
• #Number of data sources : 4606

10. MASS SPECTROMETRY
BASED PPI

11. 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

12. 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)

13. 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 speciesSpecies and fragment outcome
Fig.
• Data complexity of MS-MS / MSn spectra
• Fragment ions of “peptide versions”
• modified peptide version: Partial/full crosslinked peptide-two
still attached
• Additional PTMs

14. 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

15. 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

16. Previous cross-linkers
• Protein fragments, cross link fragments
prevents 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

17. Protein Interaction reporters (2005)
• Labile bond in the cross linker is MS
cleavable (in situ)
Tunable:
•labile bonds
•reaction groups
•affinity groups

18. 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

19. 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%

20. multiplexed FTICR-MS Spectra
• Inter cross link determination…

21. 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)

22. PIR isotope label
• PIR isotope-label differs in 6-8Da:
• relative quantitation via observed peak
intensities
→method similar to ICAT, ITRAQ

23. 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: