The post-translational modification of proteins by electrophilic oxylipids is emerging as an important mechanism that contributes to the complexity of proteomes. Enzymatic and nonenzymatic oxidation of biological lipids results in the formation of chemically diverse electrophilic carbonyl compounds, such as 2-alkenals and 4-hydroxy alkenals, epoxides and eicosanoids with reactive cyclopentenone structures. These lipoxidation products are capable of modifying proteins. Originally considered solely as markers of oxidative insult, more recently the modifications of proteins by lipid peroxidation products are being recognized as a new mechanism of cell signaling with relevance to redox homeostasis, adaptive response and inflammatory resolution. The growing interest in protein modifications by reactive oxylipid species necessitates the availability of methods that are capable of detecting, identifying and characterizing these protein adducts in biological samples with high complexity. However, the efficient analysis of these chemically diverse proteins presents a considerable analytical challenge. We first provide an introduction into the chemistry and biological relevance of the protein adduction by electrophilic lipoxidation products. We then provide an overview of tandem mass spectrometry approaches that have been developed in recent years for the interrogation of protein modifications by electrophilic oxylipid species.

Arginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many carcinomas and in the majority of sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing ASS1. As arginine-based multiagent therapies are being developed, further characterization of the changes induced by arginine starvation is needed. In order to develop a systems-level understanding of these changes, activity-based proteomic profiling (ABPP) and phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-PEG20-sensitive and resistant sarcoma cells. When integrated with metabolomic profiling, this multi-omic analysis reveals that cellular response to arginine starvation is mediated by adaptive ERK signaling and activation of the Myc-Max transcriptional network. Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by enhancing glutamine and pyruvate anaplerosis and altering lipid metabolism to recycle citrate for oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, these multi-omic approaches could provide valuable tools for evaluating response to metabolically targeted therapies.

25Protein phosphorylation is one of the most prevalent post-translational modifications found in 26 eukaryotic systems and serves as a key molecular mechanism by which protein function is 27 regulated in response to environmental stimuli. The Mut9-Like Kinases (MLKs) are a plant-specific 28 family of Ser/Thr kinases that have been linked to light, circadian, and abiotic stress signaling. 29Here we use quantitative phosphoproteomics in conjunction with global proteomic analysis to 30 explore the role of the MLKs in daily protein dynamics. In the absence of MLK family kinases, 31 proteins involved in light, circadian, and hormone signaling as well as several chromatin modifying 32 enzymes were found to have altered phosphorylation profiles. Additionally, mlk mutant seedlings 33 were found to have elevated glucosinolate accumulation and increased sensitivity to DNA 34 damage. Our analysis in combination with previously reported data supports the involvement of 35

22Arginine auxotrophy due to the silencing of argininosuccinate synthetase 1 (ASS1) occurs in many 23 cancers, especially sarcomas. Arginine deiminase (ADI-PEG20) therapy exploits this metabolic 24 vulnerability by depleting extracellular arginine, causing arginine starvation. ASS1-negative cells 25 develop resistance to ADI-PEG20 through a metabolic adaptation that includes re-expressing 26 ASS1. As arginine-based multiagent therapies are being developed, further characterization of 27 the changes induced by arginine starvation is needed. In order to develop a systems-level 28 understanding of these changes, activity-based proteomic profiling (ABPP) and 29 phosphoproteomic profiling were performed before and after ADI-PEG20 treatment in ADI-30 PEG20-sensitive and resistant sarcoma cells. When integrated with previous metabolomic 31 profiling (Kremer et al, 2017a), this multi-omic analysis reveals that cellular response to arginine 32 starvation is mediated by adaptive ERK signaling, driving a Myc-Max transcriptional network. 33Concomitantly, these data elucidate proteomic changes that facilitate oxaloacetate production by 34 enhancing glutamine and pyruvate anaplerosis, and altering lipid metabolism to recycle citrate for 35 oxidative glutaminolysis. Based on the complexity of metabolic and cellular signaling interactions, 36 these multi-omic approaches could provide valuable tools for evaluating response to metabolically 37 targeted therapies. 65invariably contribute to ABPP as well (Wolfe et al, 2013; Piazza et al, 2018a; Veyel et al, 2018). 66Ultimately, ABPP integrates multiple informative proteomic parameters and provides a broad view 67 of proteomic regulation. For example, ABPP can identify adaptive kinomic changes based on 68 either altered kinase expression or activity (Duncan et al, 2012). 69The mechanisms of developing resistance to arginine starvation in sarcomas have been 70 partially defined, and include stabilization of nuclear cMyc (Prudner et al, 2019b), and increased 71 glutamine anaplerosis in order to produce aspartate (Kremer et al, 2017a). In addition, others 72 have examined mechanisms of ASS1 re-expression (Tsai et al, 2017; Long et al, 2017) and 73Deptor regulation (Ohshima et al, 2017). However, the underlying proteomic changes that initiate 74 these events and coordinate metabolic reprogramming remain unknown. We pursued systems 75 biology profiling to understand resistance to arginine starvation, as these approaches have proven 76 effective in delineating the adaptive changes involved in highly pleiotropic phenotypes such as 77 drug resistance (Zecena et al, 2018; Galluzzi et al, 2014), Myc activation, and various metabolic 78 changes (Tomita & Kami, 2012; Schaub et al, 2018). 79To understand ADI-PEG20-resistance of ASS1-negative sarcomas at a systems level, we 80 performed multi-omic profiling using phosphoproteomics and activity-based proteomics, and 81 coupled these data with existing metabolomic analyses (Kremer et al, 2017a). ADI-PEG20-82 senstive leiomyosarcoma cells (SKLMS1) h...