ORIGINAL PAPER Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry Xiaofeng Yang • James E. Brownell • Qing Xu • Fengying Zhu • Jingya Ma • Huay-Keng Loke • Neil Rollins • Teresa A. Soucy • James J. Minissale • Michael P. Thomas • William D. Mallender • Lawrence R. Dick • Ping Li • Hua Liao Published online: 11 June 2013 Ó Springer Science+Business Media New York 2013 Abstract Ubiquitin (Ub) and ubiquitin-like (Ubl) pro- teins regulate a variety of important cellular processes by forming covalent conjugates with target proteins or lipids. Ubl conjugation is catalyzed by a cascade of proteins including activating enzymes (E1), conjugating enzymes (E2), and in many cases ligation enzymes (E3). The dis- covery of MLN4924 (Brownell et al., Mol Cell 37: 102–111, 1), an investigational small molecule that is a mechanism-based inhibitor of NEDD8-activating enzyme (NAE), reveals a promising strategy of targeting E1/Ubl pathway for therapeutic purposes. In order to better understand, the biochemical dynamics of Ubl conjugation in cells and tissues, we have developed a mass spectrom- etry-based method to quantify E1 and Ubls using isotope- labeled proteins as internal standards. Furthermore, we have used the described method to quantify levels of the covalent Nedd8-inhibitor adduct formed in MLN4924 treated cells and tissues. The Nedd8–MLN4924 adduct is a tight-binding inhibitor of NAE, and its cellular concentra- tion represents an indirect pharmacodynamic readout of NAE/Nedd8 pathway inhibition. Keywords Absolute quantification Á Mass spectrometry Á Ubiquitin (Ub) Á Ubiquitin-like proteins (Ubl) Á Ubiquitin activating enzymes (E1) Á MLN4924 Introduction Ubiquitin (Ub) is the founding member of the ubiquitin- like protein (Ubl) family that includes Nedd8, Sumo1/2/3, Fat10, Urm1, Ufm1, ISG15, ATG12, and the six human orthologs of ATG8 [2]. Ub is a 76 amino acid protein that covalently modifies other proteins, including itself, and is best known for targeting proteins for degradation by the proteasome, but also regulates protein function in a variety of other ways [3]. As a group, all Ubls similarly form covalent conjugates with proteins or, in some cases, lipids, and also share a characteristic three-dimensional fold with Ub [4]. At the amino acid sequence level, however, con- servation among Ubls varies widely [5]. Ubl conjugation pathways are vital to cellular protein homeostasis and other biological processes, including those implicated in human diseases such as neurodegeneration, autoimmunity, and cancer [6]. The importance of Ubl pathways has prompted the need for improved methods to directly characterize pathway components and substrates in cells and tissues. All Ubls are capable of forming conjugates with cellular proteins with the exception of the ATG8 orthologs which form conjugates with the lipid phosphatidylethanolamine (PE). There are eight Ubl pathway specific enzyme cas- cades that are required for conjugate formation. Each pathway requires the coordinated activity of an activating enzyme (E1), a conjugating enzyme (E2) and, in most instances, a ligase (E3) [7]. A large number of proteases, collectively referred to as deubiquitinating enzymes (DUBs), are also essential components of Ubl conjugation pathways [8]. This is because the majority of Ubls are translated as pro-proteins or as linear fusion proteins, and DUB activity is required to generate their mature forms. DUBs can also remove Ubl conjugates from substrates, and thus attenuate Ubl signaling and regenerate free Ubls. X. Yang Á J. E. Brownell Á Q. Xu Á F. Zhu Á J. Ma Á H.-K. Loke Á N. Rollins Á T. A. Soucy Á J. J. Minissale Á M. P. Thomas Á W. D. Mallender Á L. R. Dick Á P. Li Á H. Liao (&) Discovery, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA 02139, USA e-mail: hua.liao@mpi.com 123 Cell Biochem Biophys (2013) 67:139–147 DOI 10.1007/s12013-013-9625-5