Solid-Phase Compatible Silane-Based Cleavable Linker Enables Custom Isobaric Quantitative Chemoproteomics

Abstract

Mass spectrometry-based chemoproteomics has emerged as an enabling technology for functional biology and drug discovery. To address limitations of established chemoproteomics workflows, including cumbersome reagent synthesis and low throughput sample preparation, here, we established the silanebased cleavable isotopically labeled proteomics (sCIP) method. The sCIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc)protected amino acid building blocks, which enable the facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents. sCIP is compatible with both MS1-and MS2-based quantitation, and the sCIP-MS2 method is distinguished by its click-assembled isobaric tags in which the reporter group is encoded in the sCIP capture reagent and balancer in the pan cysteine-reactive probe. The sCIP-MS2 workflow streamlines sample preparation with early stage isobaric labeling and sample pooling, allowing for high coverage and increased sample throughput via customized low cost six-plex sample multiplexing. When paired with a custom FragPipe data analysis workflow and applied to cysteine-reactive fragment screens, sCIP proteomics revealed established and unprecedented cysteine-ligand pairs, including the discovery that mitochondrial uncoupling agent FCCP acts as a covalent-reversible cysteine-reactive electrophile.

Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics

“…Guided by the amine-based labeling strategy used to generate the Azido-TMT reagents 76 , we envisioned that such a reagent could be easily subjected to late-stage functionalization with commercially available activated ester reagents. Enabled by our previously described solid-phase compatible DADPS building block 75 , solid-phase peptide synthesis (SPPS) proceeded smoothly, yielding the final capture reagent (sCIP–Gly–NH 2 ) in 53% yield and high purity (Figs. 2A and S1 ).…”

“…Having demonstrated the highly efficient formation of sCIP-TMTzero, we next assessed reagent performance in chemoproteomics. We selected our established cysteine profiling workflow for benchmarking 11 , 50 , 51 , 75 , 79 – 82 . Following the workflow shown in Fig.…”

“…As our prior studies had revealed the formation of fragment ions derived from chemoproteomics-modified peptides 75 , 79 , we additionally opted to perform diagnostic ion mining analysis 83 on our sCIP-TMTzero-labeled sample. We found the TMTzero reporter ( m / z 126.1277) was the dominant ion identified in nearly 100% of modified spectra, having an average intensity >80% (Supplementary Data 1 ).…”

“…As illustrated by our own silane-based cleavable isotopically labeled proteomics (sCIP) method 75 and the recently reported azidoTMT method 76 , an alternative strategy is to introduce the isobaric label earlier in sample preparation via a fully functionalized “clickable” handle that features the built-in capacity for sample enrichment. The key advance of the sCIP platform was our fully functionalized enrichment reagents that contain biotin, a chemically cleavable dialkoxydiphenylsilane (DADPS) group, an azide for copper-catalyzed azide-alkyne cycloaddition (CuAAC or “click”) enrichment, and an isobaric label.…”

Virus-like Iron-Gold Heterogeneous Nanoparticles for Drug Target Screening

“…In recent years, chemoproteomic methods have rapidly developed and emerged as effective target screening tools. − They have made significant progress in drug target discovery and identification compared with other methods, such as substrate assays and phenotype-based screening , due to their high throughput, accuracy, and sensitivity. Notably, affinity-based proteomics , is one of the most commonly used methods for drug target identification owing to the expanded target protein range and preservation of protein’s physical and chemical properties . In affinity-based proteomics, small molecule drugs are attached to solid-phase matrices covalently or through other interaction forces .…”