Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.Proteomic technologies based on mass spectrometry (MS) have emerged as preferred components of a strategy for discovery of diagnostic, prognostic and therapeutic protein biomarkers. Because of the stochastic sampling of proteomes in unbiased analyses and the associated high false-discovery rate, tens to hundreds of potential biomarkers are often reported in discovery studies. Those few that will ultimately show sufficient sensitivity and specificity for a given medical condition must thus be culled from lengthy lists of candidates -a particularly challenging aspect of the biomarker-development pipeline and currently its main limiting step. In this context, it is highly desirable to verify, by more targeted quantitative methods, the levels of candidate biomarkers in body fluids, cells, tissues or organs from healthy individuals and affected patients in large enough sample numbers to confirm statistically relevant differences 1, 2. Verification of novel biomarkers has relied primarily on the use of sensitive, specific, high-throughput immunoassays, whose development depends critically on the availability of suitable well-characterized antibodies. However, antibody reagents of sufficient specificity and sensitivity to assay novel protein biomarkers in plasma are generally not available. The high cost and long development time required to generate high-quality immunoassay reagents, as well as technical limitations in multiplexing immunoassays for panels of biomarkers, is strong motivation to develop more straightforward quantitative approaches exploiting the sensitivity and molecular specificity of mass spectrometry.Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution (SID)-MS for direct quantification of proteins in cell lysates as well as human plasma and serum has been shown to have considerable promise 3- RESULTS Study de...

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.

Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD + -dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis. L ysine acetylation is one of the most common posttranslational modifications among cellular proteins and regulates a variety of physiological processes including enzyme activity, proteinprotein interactions, gene expression, and subcellular localization (1). Large-scale proteomic surveys have demonstrated that lysine acetylation is prevalent within mitochondria (2, 3). As the central regulators of cellular energy production, mitochondria require a coordinated response to changes in nutrient availability to respond to metabolic needs. In addition to ATP production, mitochondria are essential for regulation of fatty acid oxidation, apoptosis, and amino acid catabolism. Disruption of these processes is associated with a variety of neurodegenerative disorders and metabolic diseases (4, 5). Understanding the role of lysine acetylation in mitochondrial function will likely provide insight into altered metabolism in dysregulated or disease states.The sirtuins (SIRT1-7) are an evolutionary conserved family of NAD + -dependent deacetylases (6). SIRT3, SIRT4, and SIRT5 are major, if not exclusively, localized in the mitochondrial matrix (7,8). SIRT3 is the primary regulator of mitochondrial lysine acetylation (9), whereas SIRT5 regulates lysine malonylation and succinylation (10, 11), and SIRT4 has no well established target except weak ADP ribosyltransferase activity (12). SIRT3 is highly expressed in mitochondria-rich tissues and shows differentially regulated expression in liver and skeletal muscle in response to changes in nutrient availability such as fas...

SUMMARYTargeting defects in metabolism is an underutilized strategy for the treatment of cancer. Arginine auxotrophy resulting from the silencing of argininosuccinate synthetase 1 (ASS1) is a common metabolic alteration reported in a broad range of aggressive cancers. To assess the metabolic effects that arise from acute and chronic arginine starvation in ASS1-deficient cell lines, we performed metabolite profiling. We found that pharmacologically induced arginine depletion causes increased serine biosynthesis, glutamine anaplerosis, oxidative phosphorylation, and decreased aerobic glycolysis, effectively inhibiting the Warburg effect. The reduction of glycolysis in cells otherwise dependent on aerobic glycolysis is correlated with reduced PKM2 expression and phosphorylation and upregulation of PHGDH. Concurrent arginine deprivation and glutaminase inhibition was found to be synthetic lethal across a spectrum of ASS1-deficient tumor cell lines and is sufficient to cause in vivo tumor regression in mice. These results identify two synthetic lethal therapeutic strategies exploiting metabolic vulnerabilities of ASS1-negative cancers.

Reactive oxygen species (ROS) are both physiological intermediates in cellular signaling and mediators of oxidative stress. The cysteine-specific redox-sensitivity of proteins can shed light on how ROS are regulated and function, but low sensitivity has limited quantification of the redox state of many fundamental cellular regulators in a cellular context. Here we describe a highly sensitive and reproducible oxidation analysis approach (OxMRM) that combines protein purification, differential alkylation with stable isotopes, and multiple reaction monitoring mass spectrometry that can be applied in a targeted manner to virtually any cysteine or protein. Using this approach, we quantified the site-specific cysteine oxidation status of endogenous p53 for the first time and found that Cys182 at the dimerization interface of the DNA binding domain is particularly susceptible to diamide oxidation intracellularly. OxMRM enables analysis of sulfinic and sulfonic acid oxidation levels, which we validate by assessing the oxidation of the catalytic Cys215 of protein tyrosine phosphatase-1B under numerous oxidant conditions. OxMRM also complements unbiased redox proteomics discovery studies as a verification tool through its high sensitivity, accuracy, precision, and throughput. Molecular & Cellular Proteomics 9:1400 -1410, 2010.Oxidation of cysteine residues plays a critical role in modifying the structure and function of many proteins. Although cysteine oxidation is a tightly regulated biological process, nonenzymatic processes can contribute substantially to its levels, such as during oxidative stress. Regulatory oxidation states such as disulfide bonding and S-nitrosylation are readily modulated (1) and play an essential role in many physiological processes, including cell cycle, growth, death, and differentiation (2). In contrast, prolonged accumulation of reactive oxygen species is associated with many pathological conditions and leads to stable overoxidized states (sulfinic and sulfonic acid) that may disrupt redox regulation and protein function (3) and, in most cases, are thought to be nonregenerative.Assays capable of comprehensively assessing the dynamic changes in site-specific oxidation states are especially critical to understanding the contribution of redox status to many diseases. Numerous redox-sensitive proteins, including essential cellular regulators such as p53, have been described previously (for review, see ref. 4). However, technical factors have hampered the identification of specific site(s) of modification and characterization of their redox status in cells. Sitedirected mutagenesis is often employed to determine whether specific cysteines have redox-regulated functional roles (1), but this approach provides no information on the oxidation status of the endogenous protein. In addition, cysteine oxidation is dynamically dependent on the concentration, location, and specificity of small-molecule oxidants (5) and regulators of various antioxidant enzymes (6). Thiol pKa (7), solvent accessibility, and ...