Copper(I)-nitrene platform for chemoproteomic profiling of methionine.

Methionine plays a critical role in various biological and cell regulatory processes, making its chemoproteomic profiling indispensable for exploring its functions and potential in protein therapeutics. Building on the principle of rapid oxidation of methionine, we report Copper(I)-Nitrene Platform for robust, and selective labeling of methionine to generate stable sulfonyl sulfimide conjugates under physiological conditions. We demonstrate the versatility of this platform to label methionine in bioactive peptides, intact proteins (6.5-79.5 kDa), and proteins in complex cell lysate mixtures with varying payloads. We discover ligandable proteins and sites harboring hyperreactive methionine within the human proteome. Furthermore, this has been utilized to profile oxidation-sensitive methionine residues, which might increase our understanding of the protective role of methionine in diseases associated with elevated levels of reactive oxygen species. The Copper(I)-Nitrene Platform allows labeling methionine residues in live cancer cells, observing minimal cytotoxic effects and achieving dose-dependent labeling. Confocal imaging further reveals the spatial distribution of modified proteins within the cell membrane, cytoplasm, and nucleus, underscoring the platform's potential in profiling the cellular interactome.

Tertiary Amine Coupling by Oxidation for Selective Labeling of Dimethyl Lysine Post-Translational Modifications.

Lysine dimethylation (Kme2) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme2. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme2 peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme2 sites, in addition to previously known 5 Kme2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme2, facilitating the global identification of low-abundance Kme2-PTMs and their sites within complex cell lysate mixtures.

Bioinspired Synthesis of Allysine for Late-Stage Functionalization of Peptides.

Inspired by the enzyme lysyl oxidase, which selectively converts the side chain of lysine into allysine, an aldehyde-containing post-translational modification, we report herein the first chemical method for the synthesis of allysine by selective oxidation of dimethyl lysine. This approach is highly chemoselective for dimethyl lysine on proteins. We highlight the utility of this biomimetic approach for generating aldehydes in a variety of pharmaceutically active linear and cyclic peptides at a late stage for their diversification with various affinity and fluorescent tags. Notably, we utilized this approach for generating small-molecule aldehydes from the corresponding tertiary amines. We further demonstrated the potential of this approach in generating cellular models for studying allysine-associated diseases.

Multicomponent Oxidative Nitrile Thiazolidination Reaction for Selective Modification of N-terminal Dimethylation Posttranslational Modification.

Protein α-N-terminal dimethylation (Nme2) is an underexplored posttranslational modification (PTM) despite the increasing implications of α-N-terminal dimethylation in vital physiological and pathological processes across diverse species; thus, it is imperative to identify the sites of α-N-terminal dimethylation in the proteome. So far, only ∼300 α-N-terminal methylation sites have been discovered including mono-, di-, and tri-methylation, due to the lack of a pan-selective method for detecting α-N-terminal dimethylation. Herein, we introduce the three-component coupling reaction, oxidative nitrile thiazolidination (OxNiTha) for chemoselective modification of α-Nme2 to thiazolidine ring in the presence of selectfluor, sodium cyanide, and 1,2 aminothiols. One of the major challenges in developing a pan-specific method for the selective modification of α-Nme2 PTM is the competing reaction with dimethyl lysine (Kme2) PTM of a similar structure. We tackle this challenge by trapping nitrile-modified Nme2 with aminothiols, leading to the conversion of Nme2 to a five-membered thiazolidine ring. Surprisingly, the 1,2 aminothiol reaction with nitrile-modified Kme2 led to de-nitrilation along with the de-methylation to generate monomethyl lysine (Kme1). We demonstrated the application of OxNiTha reaction in pan-selective and robust modification of α-Nme2 in peptides and proteins to thiazolidine functionalized with varying fluorescent and affinity tags under physiological conditions. Further study with cell lysate enabled the enrichment of Nme2 PTM containing proteins.

Covalent Chemical Tools for Profiling Post-Translational Modifications.

Nature increases the functional diversity of the proteome through posttranslational modifications (PTMs); a process that involves the proteolytic processing or catalytic attachment of diverse functional groups onto proteins. These modifications modulate a host of biological activities and responses. Consequently, anomalous PTMs often correlate to a host of diseases, hence there is a need to detect these transformations, both qualitatively and quantitatively. One technique that has gained traction is the use of robust chemical strategies to label different PTMs. By utilizing the intrinsic chemical reactivity of the different chemical groups on the target amino acid residues, this strategy can facilitate the delineation of the overarching and inclusionary roles of these different modifications. Herein, we will discuss the current state of the art in post-translational modification analysis, with a direct focus on covalent chemical methods used for detecting them.