Dataset Information

Stratifying the Cell Surface Cysteinome using Two-step Enrichment Proteomics

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Leukocyte

DISEASE(S): Acute Leukemia

SUBMITTER: Tianyang Yan

LAB HEAD: Keriann Backus

PROVIDER: PXD042403 | Pride | 2024-01-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	2021-02-01-kb-70min_FAIMS_100micron_3cv_-35_-45_-55_SY52-Try-click-40-sp3-NA.pepXML	Pepxml
	2021-02-01-kb-70min_FAIMS_100micron_3cv_-35_-45_-55_SY52-Try-click-40-sp3-NA.raw	Raw
	2021-03-05-kb-70min_FAIMS_100micron_3cv_-35_-34_-55_SY54-T1.pepXML	Pepxml
	2021-03-05-kb-70min_FAIMS_100micron_3cv_-35_-34_-55_SY54-T1.raw	Raw
	2021-03-05-kb-70min_FAIMS_100micron_3cv_-35_-34_-55_SY54-T2.pepXML	Pepxml

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Publications

Defining the Cell Surface Cysteinome Using Two-Step Enrichment Proteomics.

Yan Tianyang T Boatner Lisa M LM Cui Liujuan L Tontonoz Peter J PJ Backus Keriann M KM

JACS Au 20231213 12

The plasma membrane proteome is a rich resource of functionally important 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 ena ...[more]

PMID: 38155636

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Project description:Cysteine is unique among all protein-coding amino acids owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by both a broad range of redox mechanisms and various electrophiles derived from exogenous or endogenous sources. Measuring proteomic cysteines response to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling (ABPP) based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed as QTRP (Quantitative Thiol Reactivity Profiling), that relies on the ability of a commercially available thiol-reactive probe IPM to covalently label, enrich, and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g. control vs treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics, (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also outline here how to use QTRP for measuring the changes on proteomic cysteines upon redox perturbation and for identifying targeted cysteine sites of thiol-reactive electrophiles. We anticipate that this protocol should find broad applications in both redox/chemical biology and drug discovery. The protocol for sample preparation takes 3 days, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.