Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs)

Abstract

Protein-protein interactions (PPIs) trigger a wide range of biological signaling pathways that are crucial for biomedical research and drug discovery. Various techniques have been used to study specific proteins, including affinity chromatography, activity-based probes, affinity-based probes and photo-affinity labeling (PAL). PAL has become one of the most powerful strategies to study PPIs. Traditional photocrosslinkers are used in PAL, including benzophenone, aryl azide, and diazirine. Upon photoirradiation, these photocrosslinkers (Pls) generate highly reactive species that react with adjacent molecules, resulting in a direct covalent modification. This review introduces recent examples of chemical proteomics study using PAL for PPIs.

Keywords: Aryl azide; Benzophenone; Diazirine; Photo-affinity probe; Protein-protein interaction; Quantitative proteomics.

Fig. 1

Mode of action of different photocrosslinkers

Fig. 2

Genetically encoded amino acid p-benzoyl-L-phenylalanine (Bpa) based photo-probes

Fig. 3

An in vivo covalent chemical capture and mass spectrometric-based approach for the identification of the cellular targets of transcriptional activators

Fig. 4

Design of an MP-directed activity-based probe, HxBP-Rh

Fig. 5

Mode of action and the designs of bifunctional PIP_n

Fig. 6

General structure of the lipid tools

Fig. 7

Structures of newly synthesized Lck photoligands

Fig. 8

Structure of a novel multifunctional benzophenone linker for photo-crosslinking motif and peptide stapling reagent

Fig. 9

Electrochemical approach for the PAL (a) photolithographic oxidation of as-grown BDD, (b) photolitographic decomposition of mPEG-silane chains and formation of OH-BDD patterns, (c) esterification of OH-BDD patterns with benzophenone, and (d) photoimmobilization of biomolecules on benzophenone-terminated regions

Fig. 10

Conjugation of the BP and PTR6154 using the submonomer approach

Fig. 11

Schematic representation of PPI analysis by photo-cross-linking/label transfer using cleavable benzophenone photoprobes. PDEγ/GRt interaction is presented as a model system

Fig. 12

(1) Schematic representation of the CLASPI strategy to profile H3K4Me₃ binding partners in whole-cell proteomes. a Strategy to capture proteins that recognize histone PTMs. b Structure of PHD finger of ING2 binding to a H3K4me3 peptide. c Chemical structure of probe 1. Figure reproduced from ref. [56] with permission from ACS publication

Fig. 13

a Structures of the active PAL probe 1 and the inactive probes 2–4 and their inhibitory activity against hCAII (mM) b Active/inactive PAL probes, their hCAII inhibitory potencies (IC 50), and fluorophore-conjugated click reagents for the reactions. c A efficient photo-affinity labeling approach toward identification of carbohydrate-binding proteins by using AuNP-based multivalent carbohydrate probes

Fig. 14

Structures of photo-crosslinking BODIPY (pcBD) probes

Fig. 15

The chemical structure of 3′-azibutyl-N-carbamoyl-lysine (AbK) and Synthesis of the diazirine-modified lysine

Fig. 16

(Top) A General Procedure for Protein Photo-Cross-Linking Using a Cleavable Photo-Cross-Linker. (a) In situ generation of MS-label on prey proteins by using a genetically encoded cleavable photocrosslinker. (b) Chemical design of the photocrosslinker (DiZHSeC) with transferable MS-label

Fig. 17

Chemical structures of pyrrolysine (1), AbK (2), ZLys (3), pNO₂ZLys (4), and TmdZLys (5) with DA as PL

Fig. 18

Chemical structure of GA photoprobe

Fig. 19

(left) Tag-switching strategy for the identification of target proteins by double photoreactions of a multifunctional cross-linker. Figure reproduced from ref. [67] with permission from RCS publication. (Right) A new strategy for target identification using PAL with IsoFT simplifies the identification of the target peak in both HPLC and MS analyses. Figure reproduced from ref. [68] with permission from Wiley-WCH publication

Fig. 20

Chemical structures of coumarin based PLs

Fig. 21

Photo-initiated efficient covalent coupling of diazirine modified aptamer probe with its target protein for biomarker discovery. Figure reproduced from ref. [70] with permission from RSC publication

Fig. 22

(Left) preparation and application of the HPTM dual probe, based on DNA-templated chemistry and photo-crosslinking, for the identification of HPTM reader proteins. Figure reproduced from ref. [71] with permission from Wiley-WCH publication. (Right) structure of a ^DA phosphoramidite unit

Fig. 23

([Top (a, b)]) Schematic representation of probes for affinity-based proteomic profiling; b schematic representation of affinity-based profiling of metalloproteases (Bottom) Structure of FED 1 and the probes P1 and P2

Fig. 24

(left) Chemical structures of the 3 “minimalist” linkers and 12 corresponding kinase probes (Right) second-generation approach reported in the current work, with cyclopropenes as chemically tractable tags suitable for copper-free bio-orthogonal chemistry

Fig. 25

FTO recognition mechanism of m⁶A and the design of diazirine photocrosslinking between the m⁶A-containing RNA and FTO

Fig. 26

Synthesis of 3-(m-or p-tolyl)-3-(trifluorometh-yl)-3H-diazirines

Fig. 27

(a) Schematic representation of the procedure for PAL of lectins with carbohydrate photoprobe and isolation of photo-crosslinked proteins via tandem application of SPAAC, biotin-streptavidin enrichment and a photo-release step. (b) Design of the multivalent photo-affinity glycoprobe (1) and the photo-cleavable biotin affinity tag (2). Figure reproduced from ref. [79] with permission from ScienceDirect publication

Fig. 28

(Left) Structures of lactose-based photo-affinity probes and control probes bearing TPD or alkyl diazirine groups. (Right) Synthetic plan for synthesizing clickable photo-affinity probe1 by site-selective acylation of OSW-1. MBz = 4-methoxybenzoyl

Fig. 29

a Schematic representation of bioorthogonal chemistry approach for to biomolecular interactions. b Reagents for delivering photocrosslinking functionality to azide-labeled biomolecules, including the previously reported PhosDAz and the reagent reported herein, BCN-DAz-Biotin. Figure reproduced from ref. [82] with permission from RSC publication

Fig. 30

Structures of photocrosslinking amino acids that have been incorporated into cellular proteins

Fig. 31

Schematic representation of multivalency approach

Fig. 32

Chemical structures of photo-affinity probes 1–5 and C1

Fig. 33

(SAH)-based photoreactive probes for chemical proteomic profiling of methyltransferases

Fig. 34

PAL based on Photoactivatable Isoprenoid. Figure reproduced from ref. [99] with permission from ACS publication

Fig. 35

(left) Structures of glycolipid photo-affinity probes (1,2) with corresponding control probes (inactive probes) (3,4). (Right) Structures of lactose-based photo-affinity probes

Fig. 36

(top) Schematic of photo-affinity-based target identification (ID) with different photoactivatable linkers. Each target ID probe containing a photoactivatable moiety (BP, DA and AA) which can bind to a specific set of proteins in a structure-dependent manner. Figure reproduced from ref. [102] with permission from ACS publication. (Bottom) Schematic illustration of the molecular shape-dependence of protein labeling. The flexibility of linear molecules increases the binding to various proteins. Branched molecules bind to fewer proteins than linear molecules due to their restricted conformational flexibility. Figure reproduced from ref. [103] with permission from RSC publication

Fig. 37

Photo-affinity probes with crosslinking groups attached to Ala46 a) Phenyl-azide-based ubiquitin probes b) The diazirine-based ubiquitin probes

Fig. 38

Chemical structure of novel AdoMet analogues with photo-cross-linking side chains

Fig. 39

Schematic overview of the photo-affinity PEGylation using GSH-BP

Fig. 40

schematic representation of the application of photo-affinity probes with potential photo-affinity probes for 2-oxoglutarate oxygenases incorporating 5 different photoreactive groups. Figure reproduced from ref. [107] with permission from RSC publication

Fig. 41

Photocrosslinker probes used in this study. The photoreactive group are AA, BP and DA

Fig. 42

(Left) Design of tetrazoles with variable photoactivation wavelengths. Figure reproduced from ref. [109] with permission from ScienceDirect publication. (Right) tetrazole as a New Photo-affinity Label for Drug Target Identification. Figure reproduced from ref. [110] with permission from ACS publication

Fig. 43

Structures of tetrazole-containing one- and two-photon probes based on Bodipy and Acedan dyes, respectively

Fig. 44

Schematic outlines showing typical workflows for quantitative proteomics from cells or tissues (from protein extraction, trypsin digestion and/or isotope labeling to MS analysis). Label-free quantitation individually analyzes samples and compares the data using multiple approaches like spectral counting and peak intensities. As unlabeled samples are individually analyzed in label-free workflows, the steps must be tightly controlled to avoid biasness. Conversely, labeled protein quantification is characterized by the isotopic labeling of proteins either after protein extraction or in live cell condition. Then, the labeled samples are combined and processed for quantitative analysis. The red and the green colors represent heavy and light isotopes, respectively, for differential labeling and comparison