Jason M. Held

Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.

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.

Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways

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.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Targeted Quantitation of Site-Specific Cysteine Oxidation in Endogenous Proteins Using a Differential Alkylation and Multiple Reaction Monitoring Mass Spectrometry Approach

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.

N-acylethanolamine signalling mediates the effect of diet on lifespan in Caenorhabditis elegans.

Dietary restriction is a robust means of extending adult lifespan and postponing age-related disease in many species, including yeast, nematode worms, flies and rodents. Studies of the genetic requirements for lifespan extension by dietary restriction in the nematode Caenorhabditis elegans have implicated a number of key molecules in this process, including the nutrient-sensing target of rapamycin (TOR) pathway and the Foxa transcription factor PHA-4 (ref. 7). However, little is known about the metabolic signals that coordinate the organismal response to dietary restriction and maintain homeostasis when nutrients are limited. The endocannabinoid system is an excellent candidate for such a role given its involvement in regulating nutrient intake and energy balance. Despite this, a direct role for endocannabinoid signalling in dietary restriction or lifespan determination has yet to be demonstrated, in part due to the apparent absence of endocannabinoid signalling pathways in model organisms that are amenable to lifespan analysis. N-acylethanolamines (NAEs) are lipid-derived signalling molecules, which include the mammalian endocannabinoid arachidonoyl ethanolamide. Here we identify NAEs in C. elegans, show that NAE abundance is reduced under dietary restriction and that NAE deficiency is sufficient to extend lifespan through a dietary restriction mechanism requiring PHA-4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietary-restriction-induced lifespan extension in wild-type worms, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signalling in ageing and indicates that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan.

Design, Implementation and Multisite Evaluation of a System Suitability Protocol for the Quantitative Assessment of Instrument Performance in Liquid Chromatography-Multiple Reaction Monitoring-MS (LC-MRM-MS)

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Preferentially Increased Nitration of α-Synuclein at Tyrosine-39 in a Cellular Oxidative Model of Parkinson’s Disease

Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinsons disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to controls) in levels of 3NT at Tyr-39 of alpha-synuclein in an inducible transgenic cellular model of Parkinsons disease in which monoamine oxidase B (MAO-B) is overexpressed and which emulates several features of PD. Increased nitration of Tyr-39 on endogenous alpha-synuclein via elevations in MAO-B levels could be abrogated by the addition of deprenyl, a specific MAO-B inhibitor. The increased levels of 3NT was selective for Tyr-39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr-125, Tyr-133, and Tyr-136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein.

Regulatory Control or Oxidative Damage? Proteomic Approaches to Interrogate the Role of Cysteine Oxidation Status in Biological Processes

Oxidation is a double-edged sword for cellular processes and its role in normal physiology, cancer and aging remains only partially understood. Although oxidative stress may disrupt biological function, oxidation-reduction (redox) reactions in a cell are often tightly regulated and play essential physiological roles. Cysteines lie at the interface between these extremes since the chemical properties that make specific thiols exquisitely redox-sensitive also predispose them to oxidative damage by reactive oxygen or nitrogen species during stress. Thus, these modifications can be either under reversible redox regulatory control or, alternatively, a result of reversible or irreversible oxidative damage. In either case, it has become increasingly important to assess the redox status of protein thiols since these modifications often impact such processes as catalytic activity, conformational alterations, or metal binding. To better understand the redox changes that accompany protein cysteine residues in complex biological systems, new experimental approaches have been developed to identify and characterize specific thiol modifications and/or changes in their overall redox status. In this review, we describe the recent technologies in redox proteomics that have pushed the boundaries for detecting and quantifying redox cysteine modifications in a cellular context. While there is no one-size-fits-all analytical solution, we highlight the rationale, strengths, and limitations of each technology in order to effectively apply them to specific biological questions. Several technological limitations still remain unsolved, however these approaches and future developments play an important role toward understanding the interplay between oxidative stress and redox signaling in health and disease.

Systematic mapping of posttranslational modifications in human estrogen receptor alpha, with emphasis on novel phosphorylation sites

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.

DAF-12-dependent rescue of dauer formation in Caenorhabditis elegans by (25S)-cholestenoic acid

Population density, temperature and food availability all regulate the formation of the Caenorhabditis elegans dauer larva by modulating endocrine signaling pathways. The orphan nuclear receptor DAF‐12 is pivotal for the decision to form a dauer or to undergo normal reproductive development. The DAF‐12 ligand has been predicted to be a sterol that is metabolized by DAF‐9, a cytochrome P450. Here we chemically characterize purified lipophilic nematode extracts and show that the ligand for DAF‐12 contains a carboxyl moiety and is likely to be derived from a sterol. Using a candidate ligand approach we find that the C27 bile acid cholestenoic acid (5‐cholesten‐3β‐ol‐(25S)‐carboxylic acid) promotes reproductive growth in dauer‐constitutive mutants in a daf‐9‐ and daf‐12‐dependent manner. Furthermore, we find that cholestenoic acid can act as a DAF‐12 ligand by activating DAF‐12 in a cell‐based transcription assay. Analysis of dauer‐rescuing lipophilic extracts from nematodes by gas chromatography–mass spectrometry indicates the presence of several regioisomers of cholestenoic acid that are distinct from Δ5‐cholestenoic acid and are not present in extracts from daf‐9 mutants. These data suggest that carboxylated sterols may be key determinants of life history.