Protein Modifications DOI: 10.1002/ange.201300252 A Chemical Probe for Lysine Malonylation** Xiucong Bao, Qian Zhao, Tangpo Yang, Yi Man Eva Fung,* and Xiang David Li* Protein posttranslational modifications (PTMs) play funda- mental roles in regulating normal cell physiology and disease pathogenesis. [1] Extensive studies on their cellular functions and mechanisms have advanced our understanding on some well-known PTMs such as phosphorylation, methylation, and acetylation. However, the biological roles played by many newly identified PTMs still remain poorly understood. [2] Lysine malonylation, a covalent modification of the side chain of lysine, with a malonyl group incorporated at the e- amine (Figure 1 a), was recently identified as a novel PTM using two different approaches. [3] One involved an antibody- based affinity purification of malonylated peptides in con- jugation with mass spectrometry. [3a] The other was through a careful analysis of the enzymatic activity and structural features of sirtuin 5 (Sirt5), a nicotinamide adenine dinucle- otide (NAD)-dependent hydrolase, which had been previ- ously classified as a deacetylase but was found to have much stronger activity toward malonyllysines over acetyllysines (Figure 1 a). [3b] Given the distinct chemical nature of malo- nyllysine with a negatively charged carboxylate group, it has been proposed that lysine malonylation could result in significant changes in protein structure and function. [2a,c] Characterization of the biological functions of malonyla- tion requires identification of malonylated protein substrates. Use of an anti-malonylysine antibody led to the identification of several malonylated proteins, including metabolic enzy- mes [3a] and histones, [4] which implies a potential role for this PTM in metabolic and epigenetic regulations. Although the antibody has been used to detect malonylated proteins, immuno- bloting methods are not ideal for monitoring the dynamics of malonylation. We still lack a more general and unbiased method to profile new malonylated substrates and examine their dynamic regulation. Herein, we present the development of an alkyne-functionalized chemical probe for efficient metabolic label- ing, robust fluorescent visualization, and mass- spectrometry-based identification of malony- lated proteins. Alkyne-carrying chemical probes that can be metabolically incorporated into proteins allow for subsequent copper(I) ion-catalyzed click chemistry to conjugate the labeled pro- teins with azide-fluorescent dyes or affinity purification tags. [5] They have therefore been used to detect and identify a variety of PTMs, including acetylation, [6] lipidation, [7] glycosylation, [8] and AMPylation. [9] Isotopic sodium malonate has been used in cell culture to enhance lysine malonylation in bacteria and human cells. [3a] This result indicated that malonate can be used for metabolic labeling of malonylated proteins. Inspired by these studies, we designed and synthesized a malonate analogue, 2-propargyl malonate (Mal-yne; Figure 1 b and Supporting Information, Scheme S1), as a potential chemical probe for lysine malonylation (Figure 1 c). We first examined whether Mal-yne could be metabol- ically incorporated into cellular proteins. A stock solution of Mal-yne (in PBS at pH 7.4) was used for metabolic labeling of HeLa S3 cells. After harvesting the cells, the whole-cell lysates were subjected to azide–alkyne click chemistry to conjugate the Mal-yne labeled proteins to a rhodamine dye. The labeled proteins were then resolved by SDS-PAGE and visualized by in-gel fluorescent imaging (Figure 1 c). Dose- and time-dependent analyses revealed that a wide range of proteins was labeled by Mal-yne at the optimal concentration of 10–20 mm for 4–6 hours (Supporting Information, Fig- ure S1). This concentration is comparable to that used in the Figure 1. a) The hypothesized enzymatic reactions for lysine (de)malonylation. b) Chem- ical formulas of Mal-yne and MalAM-yne. c) Strategy for detection and identification of malonylated protein substrates using chemical probes. [*] X. Bao, [+] Q. Zhao, [+] T. Yang, Dr. Y. M. E. Fung, Prof. X. D. Li Department of Chemistry, The University of Hong Kong Pokfulam Road, Hong Kong (China) E-mail: eva.fungym@hku.hk xiangli@hku.hk [ + ] These authors contributed equally to this work. [**] We thank Prof. Chi-Ming Che for generous support on the mass spectrometer, Prof. Dan Yang for advice and support, Prof. Quan Hao for useful discussions, and Di Hu for technical assistance. X.D.L. acknowledges a seed fund from The University of Hong Kong (201111159240) and Hung Hing Ying Physical Science Research Fund (20373739). Y.M.E.F. thanks the Small Project Funding from The University of Hong Kong (201109176193) and the Special Equipment Grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project Code: SEG_HKU02). Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201300252. 4983 Angew. Chem. 2013, 125, 4983 –4986  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim