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Dynamic protein post-translational methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases, including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an l-methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and in vivo to tag proteins with a biorthogonal alkyne that can be directly detected via liquid chromatography and tandem mass spectrometry (LC-MS/MS), or functionalized for subsequent selective enrichment and LC-MS/MS identification. Without enrichment, we identify known and novel lysine mono-, di-, and tripargylation, histidine propargylation, and arginine propargylation with site-specific resolution on proteins including heat shock protein HSPA8, the translational elongation factor eEF1A1, and the metabolic enzyme phosphoglycerate mutase 1, or PGAM1, for which methylation has been implicated in human disease. With enrichment, we identify 486 proteins known to be methylated and 221 proteins with novel propargylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice propargylates proteins across organ systems with blood-brain barrier penetrance and identifies site-specific propargylation in vivo with LC-MS/MS. Leveraging these pipelines to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.

Understanding protein interactions in the presence of biological metabolites is critical for unraveling biological processes and advancing therapeutic interventions. This study focuses on α,β-unsaturated carbonyls, particularly acrolein-derived protein modifications, unveiling a one-pot, four-step, selective chemistry that results in the formation of a heterocyclic α,β-unsaturated carbonyl, termed 3-formyl-3,4-dehydropiperidino (FDP), exclusively on lysine residues. Remarkably, this chemistry transforms lysine, a nucleophile, into an electrophilic warhead. We demonstrate its versatility in late-stage peptide diversification, precision protein engineering, and homogeneous protein labeling with diverse payloads. Additionally, FDP-lysine smoothly transforms into another heterocycle, 3-methylpyridinium (3-MP) lysine via deoxygenation and aromatization in reagentless conditions. This transformation facilitates late-stage peptide functionalization and homogeneous engineering of proteins, with MP-lysine acting as a mass booster. Leveraging this chemistry, we discovered hyperreactive sites responsible for acrolein-induced modification through chemoproteomic profiling of FDP- and MP-modified proteins. Our findings revealed changes in protein-protein interactions mediated by FDP-modified proteins and uncovered ∼1548 novel cross-linking partners of an FDP-modified protein.

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., (© 2024 Wiley-VCH GmbH.)

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., (© 2024. The Author(s).)

Lysine dimethylation (Kme 2 ) 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 Kme 2 . To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme 2 to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme 2 were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme 2 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 Kme 2 sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme 2 sites, in addition to previously known 5 Kme 2 sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme 2 , facilitating the global identification of low-abundance Kme 2 -PTMs and their sites within complex cell lysate mixtures.

Protein α-N-terminal dimethylation (Nme 2 ) 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-Nme 2 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-Nme 2 PTM is the competing reaction with dimethyl lysine (Kme 2 ) PTM of a similar structure. We tackle this challenge by trapping nitrile-modified Nme 2 with aminothiols, leading to the conversion of Nme 2 to a five-membered thiazolidine ring. Surprisingly, the 1,2 aminothiol reaction with nitrile-modified Kme 2 led to de-nitrilation along with the de-methylation to generate monomethyl lysine (Kme 1 ). We demonstrated the application of OxNiTha reaction in pan-selective and robust modification of α-Nme 2 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 Nme 2 PTM containing proteins.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Emenike, Nwajiobi and Raj.)