Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Acrolein as an alternative Broadly Reactive Activity-based Probe for Activity-based Protein Profiling of Cysteinome

ABSTRACT: Iodoacetamide (IAM)-based probes have established the activity-based protein profiling (ABPP) paradigm, but their reliance on the SN2 mechanism limits the labeling of all cysteine residues, highlighting the need for complementary probes based on alternative reactions. Additionally, probe design must balance between minimizing probe size and simplifying workflow complexity. As a solution to the above issues, we propose acrolein (ACR), a simple α, β-unsaturated carbonyl compound, as an alternative broadly reactive ABPP probe. ACR is commercially available, requires no chemical modification, and its aldehyde group facilitates efficient enrichment. We developed a user-friendly chemoproteomic platform for ACR, enabling the identification of over 28,000 nucleophilic residues across three cell lines. ACR demonstrated its ability to detect reactivity changes in nucleophilic residues under native and denatured conditions, and to identify known targets of entacapone. Overall, ACR represents a versatile and efficient tool for studying functional nucleophilic sites, with potential applications in drug discovery.

ORGANISM(S): Homo Sapiens

SUBMITTER: Mingliang Ye

PROVIDER: PXD063785 | iProX | Thu May 08 00:00:00 BST 2025

REPOSITORIES: iProX

ACCESS DATA

Json Xml

Similar Datasets

A General Approach for Activity-based Protein Profiling of Oxidoreductases with Redox-differentiated Diarylhalonium Warheads

Project description:Activity-based protein profiling (ABPP) is a unique proteomic tool for measuring the activity of enzymes in their cellular context, which has been well established for enzyme classes exhibiting a characteristic nucleophilic residue (e.g. hydrolases). In contrast, the enzyme class of oxidoreductases has received less attention, as its members rely mainly on cofactors instead of nucleophilic amino acid residues for catalysis. ABPP probes have been designed for specific oxidoreductase subclasses, which rely on the oxidative conversion of the probes into strong electrophiles. Here we describe the development of ABPP probes for the simultaneous labeling of various subclasses of oxidoreductases. The probe warheads are based on hypervalent diarylhalonium salts, which show unique reactivity as their activation proceeds via a reductive mechanism resulting in aryl radicals leading to covalent labeling of liver proteins at several different amino acids in close proximity to the active sites. The redox potential of the probes can be tuned by isosteric replacement varyring the halonium central atom. ABPP experiments with liver using 16 probes differing in warhead, linker, and structure revealed distinct overlapping profiles and broad substrate specificities of several probes. With their capability of multi oxidoreductase subclass labeling – including rare examples for the class of reductases – and their unique design, the herein reported probes offer new opportunities for the investigation of the “oxidoreductome” of microorganisms, plants, animal and human tissues.

2025-05-06 | PXD043873 | Pride

SITE-SPECIFIC ACTIVITY-BASED PROTEIN PROFILING USING PHOSPHONATE HANDLES

Project description:Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses IMAC-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe (ABP), enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID–ABPP (Activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated Afatinib derivative (PF-06672131). As PhosID–ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor (EGFR), which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID–ABPP is highly complementary to biotin-based enrichment strategies in activity-based protein profiling studies, with PhosID–ABPP providing the advantage of direct ABP-interaction site identification.

2023-01-10 | PXD036569 | Pride

Proteomic Profiling of Oxidative Stress Response Proteins Using Methionine Sulfoxide-Inspired ABPP Probes

Project description:Methionine oxidation is a significant signature of oxidative stress. In nature, oxidative stress from reactive oxygen species or reactive chlorine species (ROS/RCS) serves as a fundamental defense mechanism against pathogenic sources. However, previous tools for studying methionine oxidation have depended on sulfoxide reduction strategies, making them unsuitable for use under oxidative stress conditions. Moreover, no ABPP tools have been reported for studying the activity of cellular methionine sulfoxide reductases. In this paper, we present a cysteine-targeted chemical probe inspired by methionine sulfoxide for profiling oxidative stress response proteins, including methionine sulfoxide reductases. Our probe efficiently labels nucleophilic cysteines of methionine sulfoxide reductases and functions as an activity-based protein profiling tool. The probe demonstrated good reactivity and stability, making it applicable to both in vitro and in vivo experiments under oxidative stress conditions. We showed that our probe labeled oxidative stress-related proteins in E. coli under hypochlorite stress. Finally, we demonstrated that our ABPP probe offers complementary cysteine reactivity profiles compared to conventional probes, efficiently identifying proteins such as DJ-1 superfamily members that are poorly captured by iodoacetamide-alkyne. We believe that our probe can be used to reveal the biological mechanisms by which bacteria sense strong oxidants and endure oxidative stress, such as that encountered with neutrophils.

2026-02-09 | PXD067665 | Pride

Comparison of Targeted MS Methods in Activity-Based Protein Profiling 1: DDA

Project description:To examine the different mass spectrometry approaches to monitoring kinases after enrichment with desthiobiotinylating probes for activity-based protein profiling (ABPP), two experiments were performed with H1993 lung cancer cells. First, cell lysates were pre-treated with DMSO vehicle, dasatinib, or erlotinib prior to addition of the ATP probe for ABPP to compare the differences in kinase labeling associated with examples of kinase inhibitors that vary in target selectivity. Then, to examine changes in cellular signaling, H1993 cells were treated with vehicle controls, BEX-235 (PI3K inhibitor), or Crizotinib. LC-MS/MS using data dependent acquisition, data-independent acquisition (pSMART), parallel reaction monitoring, and selected reaction monitoring (or multiple reaction monitoring) mass spectrometry were used to detect and relatively quantify the desthiobiotinylated kinase peptides.

2017-11-27 | PXD006095 | Pride

Comparison of Targeted MS Methods in Activity-Based Protein Profiling 2: DIA

2017-11-27 | PXD006096 | Pride

Covalent Targeting of Splicing in T Cells

Project description:Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide chemical probe EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and availability of functional mRNA pools that can be depleted due to extensive alternative splicing. We further introduce a comprehensive list of proteins involved in splicing and leverage both cysteine- and protein-directed activity-based protein profiling (ABPP) data with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in primary human T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.

2024-10-09 | GSE279165 | GEO

Quantitative reactivity profiling of functional arginines in the human proteome

Project description:Arginine, a nucleophilic amino acid crucial for protein structure and function, plays a key role in various biological processes like enzyme catalysis and macromolecular interactions. Despite challenges in selectively targeting its less reactive guanidine group, we developed a novel probe featuring phenyl glyoxal, AP-1, which exhibited exceptional chemical selectivity and reactivity towards arginines. Utilizing activity-based protein profiling (ABPP), we explored the human proteome across four cancer cell lines, quantifying about 17,000 arginines. This analysis led us to identify several previously unreported key hyperreactive arginines, including R43 of PKM, R171 of LDHA, R172 of LDHB, R341 of CKB, R168 of EIF4A1, and R118 of FUBP1, that play crucial roles in protein functions, highlighting the importance of these residues in mediating enzyme activity or macromolecular interactions and their therapeutic potential. Notably, mutation of CKB’s R341 can inhibit cell proliferation and migration via downregulating energy supply. Additionally, we introduced ArGO-LDHA-1, a covalent inhibitor targeting hyperreactive arginines of LDHA, demonstrating covalent binding properties and potential to enhance chemotherapy efficacy. This work not only validated the significance of numerous arginines with profound biological functions but also introduced a novel, efficient platform for large-scale arginine reactivity profiling.

2024-12-24 | PXD059194 | Pride

Uncover New Reactivity of Genetically Encoded Alkyl Bromide Unnatural Amino Acids

Project description:Genetically encoded unnatural amino acids (Uaas) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These Uaas, including a series of alkyl bromide-based Uaas, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based Uaa, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

2022-04-04 | PXD031159 | Pride

ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA mass spectrometry methodsABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA mass spectrometry methods

Project description:Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling), that in combination with DIA MS methods allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day.

2022-04-04 | PXD031848 | Pride

A group competition strategy for covalent ligand discovery

Project description:As a powerful chemoproteomic tool, activity-based protein profiling (ABPP) has been extensively used for covalent ligand discovery. However, the current ABPP-based approaches are inherently based on indirect probe labeling competed by covalent ligands, and cannot directly compare the preferences of different ligands head-to-head. Herein, we report a Group Competition-based ABPP strategy (GC-ABPP) to allow the direct comparison of multiple ligands’ binding ability on a proteome-wide scale. By dividing a library of fully functionalized probes (FFPs) into different sub-groups and labeling the proteome simultaneously, the direct competition enables comparison of the labeling ability of different probes in drawing a global protein-ligand affinity metric. When it is applied to an expanded probe library, this strategy can be used iteratively to select the highest-affinity ligand toward a certain target protein in a multiple-round process. As a proof of concept, we synthesized 65 FFPs and employed the GC-ABPP to screen the ligand-protein reactivity for >6000 cysteine sites. After three rounds screening, we identified high-affinity ligands targeting BCAT2 and UGDH. Our “multiple ligands versus multiple proteins” screening paradigm demonstrates great potential for applications in covalent ligand/drug discovery.

2025-11-06 | PXD070395 |

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data