Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Yan, Tianyang; Julio, Ashley R; Villanueva, Miranda; Jones, Anthony E.; Ball, Andréa B; Boatner, Lisa; Turmon, Alexandra C.; Yen, Stephanie L.; Desai, Heta S; Divakaruni, Ajit S.; Backus, Keriann M.

doi:10.1101/2023.01.22.525042

Cited by 4 publications

(21 citation statements)

References 115 publications

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“…Exemplifying these reagents, isoTOP-ABPP utilizes isotopically differentiated tobacco etch virus (TEV)-cleavable azidobiotin peptide capture reagents synthesized through solid-phase peptide synthesis (SPPS). A +6 Da mass difference between "heavy" and "light" reagents, achieved via 13 C5, 15 N-Valine incorporation, allows for precursor-based (MS1) quantification of treatmentinduced changes to heavy-versus light-labeled peptide abundance.…”

Section: Significant Inroads Have Already Been Made Into Realizing Th...mentioning

confidence: 99%

“…Chemoproteomic studies, including our own 15,17,47,48,[53][54][55] , continue to rely on stable isotope incorporation, either through metabolic labeling or the aforementioned custom isotopically labeled capture reagents, and MS1 based quantification 10,42,[56][57][58] . These methods are widely adopted in large part due to their relatively reasonable cost, the compatibility of the resulting datasets with freely available software packages for analysis [59][60][61] , and increased data reproducibility afforded by the ability to combine 'treated' and 'control' samples early in the sample preparation workflow.…”

Section: Significant Inroads Have Already Been Made Into Realizing Th...mentioning

confidence: 99%

“…More recent work has demonstrated that quantification of cysteine chemoproteomic datasets can also be achieved using label free quantification (LFQ), including data dependent studies (DDA) that utilize FragPipe computational platform with MSFragger and IonQuant 15,62 and data independent analysis (DIA) that uses Spectronaut 63 .…”

Section: Significant Inroads Have Already Been Made Into Realizing Th...mentioning

confidence: 99%

“…Guided by our extensive fragment ion analysis, we next chose to synthesize a six-plex isobaric reagent set comprised of three custom isotopically differentiated iodoacetamide alkyne balancers (Figure 5A, Figure S12 and Figure S13), which would be paired with six isotopically labeled sCLIP reagents, which encode the dihydrooxazolium reporter ions. We opted to use combinations of three heavy isotopes, 13 C, 18 O, and 15 N to generate our six-plex panel of isotopologues (Figure 5B), following established precedent for these heavy isotopes being compatible with isobaric labeling strategies 85,86 . IAA isotopologues 23 and 24 were obtained via the respective heavy iodoacetic acid (Figure S12A).…”

Section: -Plex Isobaric Labeling Enabled By Sclipmentioning

confidence: 99%

“…Mass spectrometry-based chemoproteomics has emerged as enabling technology for functional biology and drug discovery. Showcasing this widespread utility, recent chemoproteomic studies have uncovered covalent degraders [1][2][3][4][5][6] , protein-protein interaction (PPI) modulators [7][8][9] , novel targets with anti-bacterial activity [10][11][12][13] , pinpointed redox sensitive cysteines [14][15][16][17] , and have shed light on the mode of action and off-targets of existing drugs and clinical candidates [18][19][20][21] .…”

Section: Introductionmentioning

confidence: 99%

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A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

Burton

Polasky

Shikwanna

et al. 2023

Preprint

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The human proteome harbors tens of thousands of ligandable or potentially druggable cysteine residues. Consequently, pinpointing the optimal covalent molecule for each cysteine residue is a key challenge for chemical probe and drug discovery campaigns. While chemoproteomic methods have enabled proteome-wide screens of electrophilic molecules, achieving comprehensive proteome-wide structure activity relationship (SAR) maps requires technical innovation in two key areas: (1) streamlined sample preparation workflows and (2) increased sample throughput via multiplexing. Recent inroads in the latter challenge have been made through the incorporation of isobaric tandem mass tags (TMT) into chemoproteomic workflows; the high cost and late-stage isobaric labeling collectively have, however, limited adoption of such MS2/MS3-based quantitation strategies. Here we report the silane-based Cleavable Linkers for Isotopically-labeled Proteomics (sCLIP) method, which harnesses custom isotopically labeled chemoproteomic capture reagents to simplify sample preparation and achieve low cost 6-plex isobaric multiplexing. The sCLIP method is enabled by a high yielding and scalable route to dialkoxydiphenylsilane fluorenylmethyloxycarbonyl (DADPS-Fmoc) protected amino acid building blocks, which enable facile synthesis of customizable, isotopically labeled, and chemically cleavable biotin capture reagents. Benchmarking of a panel of fully functionalized sCLIP capture reagents revealed performance comparable to established platforms. Using the diagnostic ion mining of the FragPipe computational pipeline, we identified a characteristic fragment ion with suitable intensity and specificity for tandem mass spectrometry (MS2)-based quantification. By harnessing this unprecedented gas phase chemistry, we established a cost-effective high performance 6-plex isobaric reagent set in which the mass balancer and reporter are encoded in the cysteine-capping and biotin capture reagents, respectively. Application of the sCLIP reagents to chemoproteomic analysis of electrophilic molecules uncovered 4805 total ligandable cysteines, including established and unprecedented cysteine-ligand pairs.

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Section: Significant Inroads Have Already Been Made Into Realizing Th...mentioning

confidence: 99%

Section: Significant Inroads Have Already Been Made Into Realizing Th...mentioning

confidence: 99%

Section: Significant Inroads Have Already Been Made Into Realizing Th...mentioning

confidence: 99%

Section: -Plex Isobaric Labeling Enabled By Sclipmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

Burton

Polasky

Shikwanna

et al. 2023

Preprint

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Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics

Yan,

Boatner,

Cui

et al. 2023

Preprint

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The plasma membrane proteome is a rich resource of functional and therapeutically relevant protein targets. Distinguished by high hydrophobicity, heavy glycosylation, disulfide-rich sequences, and low overall abundance, the cell surface proteome remains undersampled in established proteomic pipelines, including our own cysteine chemoproteomics platforms. Here we paired cell surface glycoprotein capture with cysteine chemoproteomics to establish a two-stage enrichment method that enables chemoproteomic profiling of cell Surface Cysteinome. Our Cys-Surf platform captures >2,800 total membrane protein cysteines in 1,046 proteins, including 1,907 residues not previously captured by bulk proteomic analysis. By pairing Cys-Surf with an isotopic chemoproteomic readout, we uncovered 821 total ligandable cysteines, including known and novel sites. Cys-Surf also robustly delineates redox-sensitive cysteines, including cysteines prone to activation-dependent changes to cysteine oxidation state and residues sensitive to addition of exogenous reductants. Exemplifying the capacity of Cys-Surf to delineate functionally important cysteines, we identified a redox sensitive cysteine in the low-density lipoprotein receptor (LDLR) that impacts both the protein localization and uptake of LDL particles. Taken together, the Cys-Surf platform, distinguished by its two-stage enrichment paradigm, represents a tailored approach to delineate the functional and therapeutic potential of the plasma membrane cysteinome.

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sCIP-ing towards streamlined chemoproteomics

Burton,

M Backus

2023

Preprint

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Mapping the ligandability or potential druggability of all proteins in the human proteome is a central goal of mass spectrometry-based chemoproteomics. Achieving this ambitious objective requires high throughput and high coverage sample preparation and LC-MS/MS analysis for hundreds to thousands of reactive compounds and chemical probes. Conducting chemoproteomic screens at this scale benefits from technical innovations that achieve increased sample throughput. Multiplexed analysis using commercially available amine-reactive isobaric reagents (e.g. tandem mass tags or TMT) is a favored strategy to decrease instrument acquisition time. These reagents are ideally suited for protein-based quantification applications, with efficient capping and pooling of peptides after sequence specific digestion. The added enrichment steps in nearly all chemoproteomic sample preparation workflows reveals a still largely untapped opportunity for isobaric labeling, namely incorporation of the TMT label into the chemoproteomic enrichment handle for early sample pooling and increased sample preparation throughput. Here we realize this vision by establishing the silane-based Cleavable Linkers for Isotopically-labeled Proteomics (sCIP)-TMT proteomic platform. sCIP-TMT pairs a custom click-compatible sCIP capture reagent that is readily functionalized in high yield with commercially available TMT tags. Synthesis and benchmarking of a 10-plex set of sCIP-TMT reveals a 1.5-fold decrease in sample preparation time together with high coverage and high accuracy quantification. By screening a focused library of cysteine-reactive electrophiles, we demonstrate the utility of sCIP-TMT for chemoproteomic target hunting, identifying 789 total liganded cysteines. Distinguished by its compatibility with established enrichment and quantification protocols, we expect sCIP-TMT will readily translate to a wide range of chemoproteomic applications.

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Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Cited by 4 publications

References 115 publications

A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

A solid-phase compatible silane-based cleavable linker enables custom isobaric quantitative chemoproteomics

Defining the Cell Surface Cysteinome using Two-step Enrichment Proteomics

sCIP-ing towards streamlined chemoproteomics

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