Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker

Abstract

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

Expanding the Genetic Code to Study Protein–Protein Interactions

“…[32] UAA 11 contains as elenium atom at the g-position within the lysine side chain. [32] UAA 11 contains as elenium atom at the g-position within the lysine side chain.…”

“…TheC hen group designed several selenium-based photocrosslinker UAAs (11-13,F igure 6) that allow separation of bait and prey protein after crosslinking and decorate the prey protein with au nique MS label for detection. [32] UAA 11 contains as elenium atom at the g-position within the lysine side chain. After successful incorporation of 11 into ab ait protein and UV irradiation, the selenium-carbon bond can undergo oxidative cleavage,l eaving the prey protein with aselenic acid moiety,which in turn can be captured by alkynecontaining dimethoxy aniline,thereby enabling facile enrichment and detection of prey proteins.…”

Photo‐crosslinking: An Emerging Chemical Tool for Investigating Molecular Networks in Live Cells

“…After oxidative cleavage of the crosslinked group, UAA‐modified residues on the prey protein can be detected by mass spectrometry. They also developed DiZASec which contains an additional alkyne handle that is transferrable onto the prey proteins and can be chemically modified after an oxidative cleavage reaction. Using this probe, UAA‐crosslinked peptides can be selectively enriched with streptavidin‐beads after biotin‐modification through a click reaction with biotin‐azide …”

Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry

“…The Chen lab developed a diazirine-based amino acid, DiZASeC (Se-(N-(2-(3-(but-3-yn-1-yl)-3H-diazirine-3-yl)ethyl)propionamide)-3-yl-homoselenocysteine, Figure 2), that, in addition to a terminal alkyne, also features a cleavable selenium-carbon bond-essentially making it a trifunctional ncAA [36]. It is based on an earlier photo-crosslinker by the same lab, which incorporates a selenium atom in the lysine side chain, which undergoes oxidative cleavage upon H 2 O 2 treatment and separates the crosslinked side chain from the POI [37,38].…”