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Quantitative analysis of discovery-based proteomic workflows now relies on high-throughput large-scale methods for identification and quantitation of proteins and post-translational modifications. Advancements in label-free quantitative techniques, using either data-dependent or data-independent mass spectrometric acquisitions, have coincided with improved instrumentation featuring greater precision, increased mass accuracy, and faster scan speeds. We recently reported on a new quantitative method called MS1 Filtering (Schilling et al. (2012) Mol. Cell. Proteomics 11, 202-214) for processing data-independent MS1 ion intensity chromatograms from peptide analytes using the Skyline software platform. In contrast, data-independent acquisitions from MS2 scans, or SWATH, can quantify all fragment ion intensities when reference spectra are available. As each SWATH acquisition cycle typically contains an MS1 scan, these two independent label-free quantitative approaches can be acquired in a single experiment. Here, we have expanded the capability of Skyline to extract both MS1 and MS2 ion intensity chromatograms from a single SWATH data-independent acquisition in an Integrated Dual Scan Analysis approach. The performance of both MS1 and MS2 data was examined in simple and complex samples using standard concentration curves. Cases of interferences in MS1 and MS2 ion intensity data were assessed, as were the differentiation and quantitation of phosphopeptide isomers in MS2 scan data. In addition, we demonstrated an approach for optimization of SWATH m/z window sizes to reduce interferences using MS1 scans as a guide. Finally, a correlation analysis was performed on both MS1 and MS2 ion intensity data obtained from SWATH acquisitions on a complex mixture using a linear model that automatically removes signals containing interferences. This work demonstrates the practical advantages of properly acquiring and processing MS1 precursor data in addition to MS2 fragment ion intensity data in a data-independent acquisition (SWATH), and provides an approach to simultaneously obtain independent measurements of relative peptide abundance from a single experiment.

Many late-onset proteotoxic diseases are accompanied by a disruption in homeostasis of metals (metallostasis) including iron, copper and zinc. Although aging is the most prominent risk factor for these disorders, the impact of aging on metallostasis and its role in proteotoxic disease remain poorly understood. Moreover, it is not clear whether a loss of metallostasis influences normal aging. We have investigated the role of metallostasis in longevity of Caenorhabditis elegans. We found that calcium, copper, iron, and manganese levels increase as a function of age, while potassium and phosphorus levels tend to decrease. Increased dietary iron significantly accelerated the age-related accumulation of insoluble protein, a molecular pathology of aging. Proteomic analysis revealed widespread effects of dietary iron in multiple organelles and tissues. Pharmacological interventions to block accumulation of specific metals attenuated many models of proteotoxicity and extended normal lifespan. Collectively, these results suggest that a loss of metallostasis with aging contributes to age-related protein aggregation.

Recent advances in commercial mass spectrometers with higher resolving power and faster scanning capabilities have expanded their functionality beyond traditional data-dependent acquisition (DDA) to targeted proteomics with higher precision and multiplexing. Using an orthogonal quadrupole time-of flight (QqTOF) LC-MS system, we investigated the feasibility of implementing large-scale targeted quantitative assays using scheduled, high resolution multiple reaction monitoring (sMRM-HR), also referred to as parallel reaction monitoring (sPRM). We assessed the selectivity and reproducibility of PRM, also referred to as parallel reaction monitoring, by measuring standard peptide concentration curves and system suitability assays. By evaluating up to 500 peptides in a single assay, the robustness and accuracy of PRM assays were compared to traditional SRM workflows on triple quadrupole instruments. The high resolution and high mass accuracy of the full scan MS/MS spectra resulted in sufficient selectivity to monitor 6-10 MS/MS fragment ions per target precursor, providing flexibility in postacquisition assay refinement and optimization. The general applicability of the sPRM workflow was assessed in complex biological samples by first targeting 532 peptide precursor ions in a yeast lysate, and then 466 peptide precursors from a previously generated candidate list of differentially expressed proteins in whole cell lysates from E. coli. Lastly, we found that sPRM assays could be rapidly and efficiently developed in Skyline from DDA libraries when acquired on the same QqTOF platform, greatly facilitating their successful implementation. These results establish a robust sPRM workflow on a QqTOF platform to rapidly transition from discovery analysis to highly multiplexed, targeted peptide quantitation.

Summary Protein acylation links energetic substrate flux with cellular adaptive responses. SIRT5 is a NAD + -dependent lysine deacylase and removes both succinyl and malonyl groups. Using affinity enrichment and label free quantitative proteomics, we characterized the SIRT5-regulated lysine malonylome in wild-type (WT) and Sirt5 −/− mice. 1,137 malonyllysine sites were identified across 430 proteins, with 183 sites (from 120 proteins) significantly increased in Sirt5 −/− animals. Pathway analysis identified glycolysis as the top SIRT5-regulated pathway. Importantly, glycolytic flux was diminished in primary hepatocytes from Sirt5 −/− compared to WT mice. Substitution of malonylated lysine residue 184 in glyceraldehyde 3-phosphate dehydrogenase with glutamic acid, a malonyllysine mimic, suppressed its enzymatic activity. Comparison with our previous reports on acylation reveals that malonylation targets a different set of proteins than acetylation and succinylation. These data demonstrate that SIRT5 is a global regulator of lysine malonylation and provide a mechanism for regulation of energetic flux through glycolysis.

SummaryReversible posttranslational modifications are emerging as critical regulators of mitochondrial proteins and metabolism. Here, we use a label-free quantitative proteomic approach to characterize the lysine succinylome in liver mitochondria and its regulation by the desuccinylase SIRT5. A total of 1,190 unique sites were identified as succinylated, and 386 sites across 140 proteins representing several metabolic pathways including β-oxidation and ketogenesis were significantly hypersuccinylated in Sirt5−/− animals. Loss of SIRT5 leads to accumulation of medium- and long-chain acylcarnitines and decreased β-hydroxybutyrate production in vivo. In addition, we demonstrate that SIRT5 regulates succinylation of the rate-limiting ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) both in vivo and in vitro. Finally, mutation of hypersuccinylated residues K83 and K310 on HMGCS2 to glutamic acid strongly inhibits enzymatic activity. Taken together, these findings establish SIRT5 as a global regulator of lysine succinylation in mitochondria and present a mechanism for inhibition of ketogenesis through HMGCS2.