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SP3-enabled rapid and high coverage chemoproteomic identification of redox-sensitive cysteines

ABSTRACT: Cysteine residues undergo various oxidative modifications, acting as key sensors of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Given that ROS and RNS have known roles in many pathophysiological processes, numerous proteome-wide strategies to profile cysteine oxidation state have emerged in the last decade. Recent advancements to traditional redox profiling methods include incorporation of costly isotopic labeling reagents to allow for more quantitative assessment of oxidation states. These methods are typically carried out by using sequential thiol capping and reduction steps in order to label redox-sensitive cysteines, often found in di-cysteine motifs (‘CXXC’ or ‘CXXXC’). Tailored, pricy algorithms are commonly used to analyze redox-profiling datasets, the majority of which cannot accurately quantify site-of-labeling in redox-motifs; moreover, accurate quantification is confounded by excess labeling reagents during sample preparation. Here, we present a low-cost redox-profiling workflow using newly synthesized isotopic reagents compatible with SP3-bead technology, termed SP3-ROx, that allows for high throughput, rapid identification of redox-sensitive cysteines. We optimize cysteine labeling quantification using the FragPipe suite, an open source GUI for MSfragger-based search algorithm. Application of SP3-ROx to naive and activated T cells identifies redox-senstive cysteines, showcasing the utility of this workflow to study biological processes.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Cell Culture, Jurkat Cell

SUBMITTER: Heta Desai

LAB HEAD: Keriann Backus

PROVIDER: PXD029500 | Pride | 2022-03-02

REPOSITORIES: Pride

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SP3-enabled rapid and high coverage chemoproteomic identification of redox-sensitive cysteines

Project description:Cysteine residues undergo various oxidative modifications, acting as key sensors of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Given that ROS and RNS have known roles in many pathophysiological processes, numerous proteome-wide strategies to profile cysteine oxidation state have emerged in the last decade. Recent advancements to traditional redox profiling methods include incorporation of costly isotopic labeling reagents to allow for more quantitative assessment of oxidation states. These methods are typically carried out by using sequential thiol capping and reduction steps in order to label redox-sensitive cysteines, often found in di-cysteine motifs (‘CXXC’ or ‘CXXXC’). Tailored, pricy algorithms are commonly used to analyze redox-profiling datasets, the majority of which cannot accurately quantify site-of-labeling in redox-motifs; moreover, accurate quantification is confounded by excess labeling reagents during sample preparation. Here, we present a low-cost redox-profiling workflow using newly synthesized isotopic reagents compatible with SP3-bead technology, termed SP3-ROx, that allows for high throughput, rapid identification of redox-sensitive cysteines. We optimize cysteine labeling quantification using the FragPipe suite, an open source GUI for MSfragger-based search algorithm. Application of SP3-ROx to naive and activated T cells identifies redox-senstive cysteines, showcasing the utility of this workflow to study biological processes.

2022-03-02 | PXD031647 | Pride

Stratifying the Cell Surface Cysteinome using Two-step Enrichment Proteomics

Project description:Cell surface cysteines represent an attractive class of residues for chemoproteomic studies due to their accessibility towards drugs and key roles they play in the structure and function of most human proteins. The redox sensitivity of cysteines also makes cell surface cysteines potential markers for cellular activities with altered redox states. However, cell surface cysteines are underrepresented in most of the cysteine proteomic dataset. While many mass spectrometry (MS) based techniques have been developed to enrich membrane proteins, current studies lack methods that can specifically inventory cysteines on cell surfaces. Here, we developed a novel dual enrichment method to achieve chemoproteomic profiling of cell Surface Cysteines - “Cys-Surf''. Combining cell surface capture (CSC) biotinylation and cysteine chemoproteomic biotinylation, this two-step biotinylation platform achieves identification of more than 1,900 cysteines with a specificity of about 50.0% in cell surface localization. In addition, Cys-Surf achieved quantitative analysis of oxidation states of cell surface cysteines, which reflect a completely different profile compared to that of the whole cysteinome. Redox sensitive cell surface cysteines were identified by applying reducing reagents and during T cell activation. Cys-Surf is also compatible with competitive small-molecule screening by isotopic tandem orthogonal activity-based protein profiling (isoTOP-ABPP) to evaluate the ligandability of cell surface cysteines. Altogether, these findings establish a platform that enables redox and ligandability analysis of the cell surface cysteinome and sheds light on future functional studies and drug discovery efforts targeting cell surface cysteines.

2024-01-26 | PXD042403 | Pride

Identification of thioredoxin-dependent regulatory cysteines in Chlamydomonas reinhardtii using the reductome approach.

Project description:Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms. In this context, the small ubiquitous oxido-reductase thioredoxin (Trx) plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathionylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii while much less is known about the thioredoxin interactome, called thioredoxome hereafter, in this photosynthetic model organism. Using a quantitative large scale proteomic analysis based on the in vitro reconstitution of the enzymatic thioredoxin system (NADPH, NTR, Trx) within an acellular extract and followed by an isotopic labeling with cleavable Isotopic-Coded Affinity Tag reagents, we identified 1052 Trx-targeted cysteines spread over 602 proteins. These Trx-targets participate in a wide variety of metabolic pathways and cellular processes. This approach, combined with the affinity purification approach study (PXD006097) broadens not only the redox regulation to new enzymes involved in metabolic pathways already known to be regulated by thioredoxins (carbon, lipid and energy metabolism) but also shed light on cellular processes for which data supporting a putative redox regulation in these processes are scarce (aromatic amino-acid biosynthesis, nuclear transport,…). Moreover, by comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteines residues.

2017-07-28 | PXD006116 | Pride

S-bacillithiolation of the OhrR repressor and the methionine synthase MetE protect against hypochlorite stress in Bacillus subtilis

Project description:Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In B. subtilis, the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate) termed as S-bacillithiolation. We have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid (NaOCl), the active component of house-hold bleach. The OhrR-controlled peroxiredoxin OhrA was most strongly up-regulated by NaOCl stress and conferred specific protection against NaOCl. Inactivation of the OhrR repressor was caused by S-bacillithiolation of the redox-sensing Cys15 residue in response to NaOCl. Two cobalamin-independent methionine synthases MetE and YxjG were identified as S-bacillithiolated at their essential active site cysteines resulting in hypochlorite-induced methionine limitation. In summary, our studies show that S-bacillithiolation of OhrR and the methionine synthases is the major mechanism in protection against hypochlorite stress in B. subtilis. The B. subtilis 168 wild type strain was grown in minimal medium to OD500 of 0.4 and harvested before and 10 minutes after exposure to 50 µM NaOCl. Microarray hybridizations were performed in triplicate using RNA isolated from independent cultures.

2011-07-04 | E-GEOD-27637 | biostudies-arrayexpress

Rapid and high coverage profile of human cysteineome-Figure 2

Project description:Cysteines play important roles in many biological activities, including protein folding, aging, signaling and etc. To identify and quantitatively profile the oxidation and reactivity of cysteines, cysteine-reactive compounds have been applied to covalently label and enrich the cysteines. And with mass spectrometry (MS), thousands of cysteines can be profiled at the same time with decoration of functional groups. However, with all the technology and methods developed to characterize cysteines, current datasets have only uncovered about 5% of the total cysteinome. Here we show a comprehensive and high coverage profile of more than 30,000 cysteines. With incorporation of new techniques - single-pot, solid phase-enhanced sample-preparation (SP3) and High-field asymmetric waveform ion mobility spectrometry (FAIMS), we have successfully identified as many as 14,306 cystienes in a single-shot experiment. With cysteine profiles in different cell lines, proteases and fractionations, our results have successfully expanded the cysteine library to a great extent.

2021-02-03 | PXD023057 | Pride

Rapid and high coverage profile of human cysteineome-Figure 1

2021-02-03 | PXD023056 | Pride

Rapid and high coverage profile of human cysteineome

2021-02-03 | PXD023059 | Pride

Rapid and high coverage profile of human cysteineome

2021-02-03 | PXD023060 | Pride

Rapid and high coverage profile of human cysteineome-Figure 6

2021-02-03 | PXD023058 | Pride

Identification of targets of the protein disulfide reductase thioredoxin

Project description:Identification of targets of the protein disulfide reductase thioredoxin using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and thiol specific differential labeling with isotope-coded affinity tags (ICAT). Reduction of specific target disulfides is quantified by measuring ratios of cysteine residues labeled with the heavy (13C) and light (12C) ICAT reagents in peptides derived from tryptic digests of Trx-treated and non-treated samples. Keywords: protein, LC-MS/MS, ICAT

2008-11-20 | GSE11650 | GEO

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