Maleimide-Based Chemical Proteomics for Quantitative Analysis of Cysteine Reactivity

Abstract

Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a mediator against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of cysteine oxidation in understanding biological stress response, cysteine sites most reactive toward ROS remain largely unknown and are a major analytical challenge. Herein, a chemical proteomic method to quantify site-specific cysteine reactivity using a maleimide-activated, thiol-reactive probe (N-propargylmaleimide, NPM) is described. Implementation of a gel-based approach via conjugation of rhodamine-azide to NPM-labeled cysteine residues by copper-catalyzed azide−alkyne cycloaddition (CuAAC) click chemistry allowed simple and highly sensitive fluorescence profiling. Relative quantification of >1500 unique cysteine sites from greater than 800 proteins was achieved by conjugating dialkoxydiphenylsilane (DADPS) biotin-azide by the CuAAC reaction and subsequently performing biotin−streptavidin affinity purification and mass-spectrometry-based proteomics. Taken together, this work defines a novel role for the NPM probe in chemical proteomics and presents a robust method for determination of cysteine reactivity during oxidative stress response.

Proteomic Approaches to Study Cysteine Oxidation: Applications in Neurodegenerative Diseases

“…Various probes recently introduced (summarized in Figure 5C ) include (i) IA-alkyne based probes such as chloropyridine (RB2) and fluoronitrobenzene (ERW3) ( Shannon et al, 2014 ), a photocaged bromomethyl ketone (CBK) ( Abo and Weerapana, 2015 ), iodomethyl ketone (CIK4), ethynyl benziodoxolone (EBX) ( Abo et al, 2017 ), and (ii) light and heavy isotopic IA-alkyne tags ( Abo et al, 2018 ), (iii) thiol-reactive probe ( N -propargylmaleimide, NPM) ( McConnell et al, 2020 ).…”

Improving the Identification Coverage of Protein Interactome by Enhancing the Click Chemistry-based Cross-linking Enrichment Efficiency

“…Click chemistry reaction with the features of bio-orthogonality, quick reaction speed and great specificity, has been successfully applied to activity-based protein profiling, enzyme-inhibitors screening and protein labeling in proteomic analysis [15][16][17][18]. To remove biotin moiety from the enriched peptides for MS acquisition with good compatibility and deep coverage, different cleavable azide-biotin reagents, including acid-, reduction-and photo-cleavable tags were developed and used [11,19,20]. The current well-established protocols of click chemistry for proteomics analysis were mainly at protein level.…”

Redox Proteomic Analysis Reveals Microwave-Induced Oxidation Modifications of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix)

“…Recently, thiol redox proteomic tools have been used to interrogate the cysteine redox state in biological processes; , the specific cysteine residue sites in proteins could be identified by using high-resolution mass spectrometry (MS)­methods with the trap of alkylating agents . Several alkylating agents have been used to label reductive thiols in proteomes such as iodoacetamide (IAM) and N -ethylmaleimide; however, the reaction rate with protein thiols differs according to the environmental conditions. , A proteomic analysis carried out by Lu et al revealed that cysteine at the head of myosin is inclined to form sulfinic and sulfonic acids, while the cysteine site at the tail of myosin easily generates S–S under the same conditions. By using liquid chromatography–tandem MS (LC–MS/MS) for proteomic identification, Fu et al also reported that myosin and actin, which contain a high content of free sulfhydryl groups, were readily oxidized to form S–S by cross-linking in an in vitro oxidizing environment consisting of FeCl 3 , ascorbic acid, and hydrogen peroxide.…”

“…33 Several alkylating agents have been used to label reductive thiols in proteomes such as iodoacetamide (IAM) and N-ethylmaleimide; however, the reaction rate with protein thiols differs according to the environmental conditions. 33,34 A proteomic analysis carried out by Lu et al 35 revealed that cysteine at the head of myosin is inclined to form sulfinic and sulfonic acids, while the cysteine site at the tail of myosin easily generates S−S under the same conditions. By using liquid chromatography−tandem MS (LC− MS/MS) for proteomic identification, Fu et al 36 also reported that myosin and actin, which contain a high content of free sulfhydryl groups, were readily oxidized to form S−S by crosslinking in an in vitro oxidizing environment consisting of FeCl 3 , ascorbic acid, and hydrogen peroxide.…”