Traceless Cleavage of Protein–Biotin Conjugates under Biologically Compatible Conditions

Abstract Biotinylation of amines is widely used to conjugate biomolecules, but either the resulting label is non‐removable or its removal leaves a tag on the molecule of interest, thus affecting downstream processes. We present here a set of reagents (RevAmines) that allow traceless, reversible biotinylation under biologically compatible, mild conditions. Release following avidin‐based capture is achieved through the cleavage of a (2‐(alkylsulfonyl)ethyl) carbamate linker under mild conditions (200 mm ammonium bicarbonate, pH 8, 16–24 h, room temperature) that regenerates the unmodified amine. The capture and release of biotinylated proteins and peptides from neutravidin, fluorescent labelling through reversible biotinylation at the cell surface and the selective enrichment of proteins from bacterial periplasm are demonstrated. The tags are easily prepared, stable and offer the potential for future application in proteomics, activity‐based protein profiling, affinity chromatography and bio‐molecule tagging and purification.

Biotinylation of amines is widely used to conjugateb iomolecules, but either the resulting label is non-removable or its removal leaves atag on the molecule of interest, thus affecting downstream processes. We present here aset of reagents (Rev-Amines) that allow traceless,r eversibleb iotinylation under biologicallyc ompatible, mild conditions. Release followinga vidinbased capturei sa chieved throught he cleavage of a( 2-(alkylsulfonyl)ethyl) carbamate linker under mild conditions (200 mm ammoniumb icarbonate, pH 8, 16-24 h, room temperature) that regenerates the unmodifieda mine. The capture and releaseo fb iotinylated proteins and peptides from neutravidin, fluorescent labelling through reversible biotinylation at the cell surface and the selectivee nrichment of proteins from bacterialp eriplasm are demonstrated. Thet ags are easily prepared, stable and offer the potential for future applicationi n proteomics, activity-basedp rotein profiling, affinity chromatography andb io-molecule tagging and purification.
Herein we describe the synthesis of sulfonyl-based reversible amine (or "RevAmine") biotinylation reagents and demonstrate how they are capable of facile functionalization of proteinaceous amines with subsequent tracelessc leavage triggered under biologically compatible, basic conditions (pH % 8). Sulfonyl-triggerede limination reactions have been reported (e.g., amine protection, [19] protein-amine [20a,b] and polypeptide-oligonucleotide [21] crosslinking, polypeptide synthesis [22] and the slow release of drug molecules [23] ), but not under biologically compatible conditions. Therefore sulfonyl-triggerede liminations have yet to find wider application in biology and more specifically in reversible biotinylation chemistry.
Bovines erum albumin( BSA) was chosen as at est substrate to evaluate the potential for protein biotinylation. The reaction of BSA with 4 (4:BSA 20:1, PBS, 1h,r oom temperature) was examined by MALDI, which revealed that approximately eight biotin groups were incorporated per BSA ( Figure S8). Biotinylated BSAw as captured by incubation with neutravidin beads. No BSA release was observed by PAGE during elutionw ith PBS (pH 7.4), whereas elutiono fr egenerated, unmodified BSA occurredu nder ar ange of mild conditions (100 mm ammonium bicarbonate, pH 8.0, 8.5, or 9.0 as well as 0.1 %a mmonium hydroxide; Figure S9).
It should be noted that during prolonged tag-cleavage of both 4 and 7 from test peptides,m editated by ammonium bicarbonate, we observed the formation of severalm inor byproducts (Figure S10, centre). MSa nalysis ( Figure S6) suggested that these had arisen from the formation during tag cleavage of by-product vinyl-sulfones,w hich could possibly undergo subsequent nonselective Michael addition chemistry with the released peptide. The addition of dithiothreitol (DTT), as an in situ nucleophilic scavenger of the vinyl-sulfone, resulted in as ignificant reduction in by-product formation ( Figure S10, top) and is recommended when performing cleavage experiments. [20a, 24] The usefulness of tag 7 was then demonstrated in as eries of tagging, avidin capture and biocompatible release experiments (in the presence of DTT) with three small protein targets,n uclease B( Bacillusl icheniformis), cytochrome c (bovine) and apomyoglobin( equine). Ta gging (tenfold excess of 7 over protein, 1h,r oom temperature) showedad egree of substrate dependence,w ithi ncorporation of up to five biotin moieties under these conditions. The desalted, biotinylated proteins were captured on avidin beads, andt he unmodified proteins were recovered by using ammonium bicarbonate (pH 8, 24 h, room temperature). HRMS measurements of all three eluted proteins gave excellent agreement with both the measured and predicted unmodified masses (Figures S11-S13).
As af inal exemplar of the use of 7,w ed ecided upon a proteomic investigation of cell-wall-associatedp roteins in Es-cherichiac oli. [25] Porous outer-membrane mutant E. coli cells (amiABC)w eret reated with 7,f ollowedb yc ell-lysis and incubation with neutravidinb eads to capture biotinylated proteins. Unbound proteins were removed by washing the beads with lysate buffer,t hen PBS (pH 7.4). Treatment with 100 mm ammoniumb icarbonate, pH 9, overnight at 4 8C, triggered traceless linker cleavage, thus allowing elutiono ft he previously bound,b iotinylated proteins.T he protein eluate was then subjected to trypsin digestion andp roteomic analysisb yL C/MS/ MS and identification with Mascot software (Matrix Science, London).D ue to the traceless nature of the cleavage reaction, no residual mass modifications in the softwares earch were necessary to allow peptidei dentification [26] (Tables S1 and S2). After subtraction of nonspecific IDs, the 64 proteins identified in the surface-tagging experiment (TableS3) weres ubmitted to aP ANTHER over-representation test search (http://pantherdb.org/webservices/go/overrep.jsp) [27] against an E. coli background reference data set, with "cellularcomponent" as the selected enrichmentt erm. Of the proteins remaining following subtraction,t he highest-scoring enriched gene ontology terms shared by the genes on our list compared to the background distribution of annotations were:" outer membrane-bounded periplasmic space", "externale ncapsulating structure"a nd "cell envelope". Am ore than fivefold selectivee nrichment of proteins from the periplasm hadt aken place ( Figure S14).
In summary,w eh ave outlined the basis for as eries of biocompatible RevAmine tags that are synthetically accessible and are stable under neutrala nd acidic conditions, but that undergo traceless cleavage to regenerate the parent amine in the presence of aw eak base. We have shownt hat RevAminetagged proteins and peptides can undergo facile base-mediated capture-release affinity purification on neutravidin solid supports and we have demonstrated the utility of such reagentsi nc ell-surface labelling andi np roteomic analysis. Owingt ot heir biocompatible traceless-cleavage chemistry,w e envisage numerous applications of RevAmine reagents in areas such as proteomics, activity-based protein profiling, modifiable affinity chromatography supports, biomolecule tagging and biomolecule purification.