Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level

Kozhinov, Anton N.; Johnson, Alex; Nagornov, Konstantin O.; Stadlmeier, Michael; Martin, Warham Lance; Dayon, Loı̈c; Corthésy, John; Wühr, Martin; Tsybin, Yury O.

doi:10.1021/acs.analchem.2c04742

Cited by 8 publications

(6 citation statements)

References 43 publications

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“…To address this limitation, here, we implemented acquisition using a FAIMS device to reduce ratio compression. In future generation sCIP reagents, we plan to incorporate compatibility with synchronous precursor selection-real time search-LC–MS/MS/MS (SPS-RTS-MS3) and increased multiplexing capacity through alternative isobaric labeling strategies, including, for example, those that eliminate ratio compression. – When paired with off-line sample fractionation and automated sample preparation workflows, we expect to continue to streamline the throughput, coverage, and accuracy of quantitative cysteine chemoproteomics.…”

Section: Discussionmentioning

confidence: 99%

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

Burton,

Polasky,

Shikwana

et al. 2023

J. Am. Chem. Soc.

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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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Burton,

Polasky,

Shikwana

et al. 2023

J. Am. Chem. Soc.

View full text Add to dashboard Cite

show abstract

“…To address this limitation, here we implemented acquisition using a FAIMS device to reduce ratio compression. In future generation sCLIP reagents, we plan to incorporate compatibility with synchronous precursor selection-real time search-LC-MS/MS/MS (SPS-RTS-MS3) 111 and increased multiplexing capacity through alternative isobaric labeling strategies, including for example those that eliminate ratio compression [112][113][114] . When paired with off-line sample fractionation and automated sample preparation workflows, we expect to continue to streamline the throughput, coverage, and accuracy of quantitative cysteine chemoproteomics.…”

Section: Discussionmentioning

confidence: 99%

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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“…To address this limitation, here we implemented acquisition using a FAIMS device to reduce ratio compression. In future generation sCLIP reagents, we plan to incorporate compatibility with synchronous precursor selection-real time search-LC-MS/MS/MS (SPS-RTS-MS3) 112 and increased multiplexing capacity through alternative isobaric labeling strategies, including for example those that eliminate ratio compression [113][114][115] . When paired with off-line sample fractionation and automated sample preparation workflows, we expect to continue to streamline the throughput, coverage, and accuracy of quantitative cysteine chemoproteomics.…”

Section: Discussionmentioning

confidence: 99%

Enhancing multiplexed cysteine chemoproteomics by uniting FragPipe with solid-phase compatible dialkoxydiphenylsilane reagents

Burton

Polasky

Shikwanna

et al. 2023

Preprint

View full text Add to dashboard Cite

The human proteome harbors tens of thousands of ligandable or potentially druggable cysteine residues. Consequently, pinpointing the optimal covalent molecule for each cysteine residue represents an exciting means to close the druggability gap, namely the ~96% of human proteins not yet targeted by an FDA approved drug. Realizing the full therapeutic potential of the cysteineome will require comprehensive proteome-wide cysteine-compound structure activity relationship (SAR) analysis. While mass spectrometry-based chemoproteomic platforms have made significant inroads into this challenge, achieving comprehensive cysteine-SAR necessitates technical innovation in two key areas: (1) streamlined sample preparation workflows and (2) high throughput and high coverage data acquisition. Here we report the silane-based Cleavable Linkers for Isotopically labeled Proteomics (sCLIP) method. sCLIP streamlines sample preparation with unparalleled early-stage isobaric labeling and sample pooling, allowing for high coverage and increased sample throughput via customized low cost 6-plex sample multiplexing. The sCLIP method is distinguished by its unprecedented click-assembled isobaric tags, in which the reporter group is encoded in the sCLIP capture reagent and balancer in the pan cysteine-reactive probe. When paired with a custom FragPipe data analysis workflow and applied to cysteine-reactive fragment screens, sCLIP proteomics revealed established and unprecedented cysteine-ligand pairs, including the discovery that the mitochondrial uncoupling agent FCCP acts as a covalent-reversible cysteine-reactive electrophile.

show abstract

Super-Resolution Mass Spectrometry Enables Rapid, Accurate, and Highly Multiplexed Proteomics at the MS2 Level

Cited by 8 publications

References 43 publications

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

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

Enhancing multiplexed cysteine chemoproteomics by uniting FragPipe with solid-phase compatible dialkoxydiphenylsilane reagents

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