[Show abstract][Hide abstract] ABSTRACT: We describe the use of a targeted proteomics approach, Selected Reaction Monitoring (SRM) mass spectrometry, to detect and assess RNAi-mediated depletion or 'knockdown' of specific proteins from human cells and from Drosophila flies. This label-free approach does not require any specific reagents to confirm the depletion of RNAi target protein(s) in unfractionated cell or whole organism extracts. The protocol described here is general, can be developed rapidly and can be multiplexed to detect and measure multiple proteins at once. Furthermore, the methodology can be extended to any tandem mass spectrometer - making it widely accessible. This methodology will be applicable to a wide range of basic science and clinical questions where RNAi-mediated protein depletion needs to be verified, or where differences in relative abundance of target proteins need to be rapidly assessed between samples.
Journal of Proteome Research 05/2013; · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple reaction monitoring (MRM) mass spectrometry (MS) 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 eleven laboratories on a total of fifteen different instruments, enabled early diagnoses of LC and MS anomalies that indicated sub-optimal 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 (CV) <0.15, peak width CV <0.15, standard deviation of RT <0.15 min (9 sec) and the RT drift <0.5min (30 sec). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the utility and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to insure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread utilization of MRM-MS technology by the basic biomedical and clinical laboratory research communities.
[Show abstract][Hide abstract] ABSTRACT: We report a method to measure in vivo turnover of four proteins from sequential tracheal aspirates obtained from human newborn infants with respiratory distress syndrome using targeted proteomics. We detected enrichment for all targeted proteins approximately 3 h from the start of infusion of [5,5,5-(2)H(3)] leucine, secretion times that varied from 1.2 to 2.5 h, and half lives that ranged between 10 and 21 h. Complement factor B, a component of the alternative pathway of complement activation, had an approximately twofold-longer half-life than the other three proteins. In addition, the kinetics of mature and carboxy-terminal tryptic peptides from the same protein (surfactant protein B) were not statistically different (p = 0.49).
Analytical and Bioanalytical Chemistry 04/2012; 403(8):2397-402. · 3.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Software advancements in the last several years have had a significant impact on proteomics from method development to data analysis. Herein, we detail a method, which uses our in-house developed software tool termed Skyline, for empirical refinement of candidate peptides from targeted proteins. The method consists of four main steps from generation of a testable hypothesis, method development, peptide refinement, to peptide validation. The ultimate goal is to identify the best performing peptide in terms of ionization efficiency, reproducibility, specificity, and chromatographic characteristics to monitor as a proxy for protein abundance. It is important to emphasize that this method allows the user to perform this refinement procedure in the sample matrix and organism of interest with the instrumentation available. Finally, the method is demonstrated in a case study to determine the best peptide to monitor the abundance of surfactant protein B in lung aspirates.
[Show abstract][Hide abstract] ABSTRACT: The goal of many shotgun proteomics experiments is to determine the protein complement of a complex biological mixture. For many mixtures, most methodological approaches fall significantly short of this goal. Existing solutions to this problem typically subdivide the task into two stages: first identifying a collection of peptides with a low false discovery rate and then inferring from the peptides a corresponding set of proteins. In contrast, we formulate the protein identification problem as a single optimization problem, which we solve using machine learning methods. This approach is motivated by the observation that the peptide and protein level tasks are cooperative, and the solution to each can be improved by using information about the solution to the other. The resulting algorithm directly controls the relevant error rate, can incorporate a wide variety of evidence and, for complex samples, provides 18-34% more protein identifications than the current state of the art approaches.
[Show abstract][Hide abstract] ABSTRACT: There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS.
[Show abstract][Hide abstract] ABSTRACT: Proteomics experiments based on Selected Reaction Monitoring (SRM, also referred to as Multiple Reaction Monitoring or MRM) are being used to target large numbers of protein candidates in complex mixtures. At present, instrument parameters are often optimized for each peptide, a time and resource intensive process. Large SRM experiments are greatly facilitated by having the ability to predict MS instrument parameters that work well with the broad diversity of peptides they target. For this reason, we investigated the impact of using simple linear equations to predict the collision energy (CE) on peptide signal intensity and compared it with the empirical optimization of the CE for each peptide and transition individually. Using optimized linear equations, the difference between predicted and empirically derived CE values was found to be an average gain of only 7.8% of total peak area. We also found that existing commonly used linear equations fall short of their potential, and should be recalculated for each charge state and when introducing new instrument platforms. We provide a fully automated pipeline for calculating these equations and individually optimizing CE of each transition on SRM instruments from Agilent, Applied Biosystems, Thermo-Scientific and Waters in the open source Skyline software tool ( http://proteome.gs.washington.edu/software/skyline ).
[Show abstract][Hide abstract] ABSTRACT: SUMMARY: Skyline is a Windows client application for targeted proteomics method creation and quantitative data analysis. It is open source and freely available for academic and commercial use. The Skyline user interface simplifies the development of mass spectrometer methods and the analysis of data from targeted proteomics experiments performed using selected reaction monitoring (SRM). Skyline supports using and creating MS/MS spectral libraries from a wide variety of sources to choose SRM filters and verify results based on previously observed ion trap data. Skyline exports transition lists to and imports the native output files from Agilent, Applied Biosystems, Thermo Fisher Scientific and Waters triple quadrupole instruments, seamlessly connecting mass spectrometer output back to the experimental design document. The fast and compact Skyline file format is easily shared, even for experiments requiring many sample injections. A rich array of graphs displays results and provides powerful tools for inspecting data integrity as data are acquired, helping instrument operators to identify problems early. The Skyline dynamic report designer exports tabular data from the Skyline document model for in-depth analysis with common statistical tools. AVAILABILITY: Single-click, self-updating web installation is available at http://proteome.gs.washington.edu/software/skyline. This web site also provides access to instructional videos, a support board, an issues list and a link to the source code project.
[Show abstract][Hide abstract] ABSTRACT: We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.
[Show abstract][Hide abstract] ABSTRACT: Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%.
We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin-nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%.
This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species-related cardiovascular diseases.
[Show abstract][Hide abstract] ABSTRACT: Selected reaction monitoring (SRM) is a powerful tandem mass spectrometry method that can be used to monitor target peptides within a complex protein digest. The specificity and sensitivity of the approach, as well as its capability to multiplex the measurement of many analytes in parallel, has made it a technology of particular promise for hypothesis driven proteomics. An underappreciated step in the development of an assay to measure many peptides in parallel is the time and effort necessary to establish a usable assay. Here we report the use of shotgun proteomics data to expedite the selection of SRM transitions for target peptides of interest. The use of tandem mass spectrometry data acquired on an LTQ ion trap mass spectrometer can accurately predict which fragment ions will produce the greatest signal in an SRM assay using a triple quadrupole mass spectrometer. Furthermore, we present a scoring routine that can compare the targeted SRM chromatogram data with an MS/MS spectrum acquired by data-dependent acquisition and stored in a library. This scoring routine is invaluable in determining which signal in the chromatogram from a complex mixture best represents the target peptide. These algorithmic developments have been implemented in a software package that is available from the authors upon request.
Journal of Proteome Research 04/2009; 8(6):2733-9. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Werner syndrome (WS) is an autosomal recessive progeroid syndrome caused by
mutations in the Werner (Wrn) gene. WS patients have increased incidence of a
number of chronic conditions including insulin resistance and type 2 diabetes.
Since ingestion of foods that are high in fat and sugar is associated with
increased incidence of diabetes, we examined if Wrn mutations might affect
metabolic response to a diabetogenic diet. Four-month-old mice with a null
mutation for the Wrn gene were fed a diet consisting of 36% fat (lard), 33% table
sugar, and 20% protein plus balanced vitamins and minerals. Wrn null mice had
significantly increased body weights, increased serum insulin levels, impaired
glucose tolerance, and insulin resistance during 4 months of eating the
diabetogenic diet. Diffuse fatty infiltration of the liver and pancreatic islet
hyperplasia was characteristic morphological features. These observations suggest
that Wrn null mice have impaired glucose homeostasis and fat metabolism, and may
be a useful model to investigate metabolic conditions associated with aging.
Mechanisms of Ageing and Development 01/2007; 129(4):201. · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Overview Purpose: To demonstrate a laboratory and informatics workflow from discovery to targeted measurement Methods: Data were acquired using high-resolution LC-MS/MS using hybrid linear ion trap (Orbitrap TM) and triple quadrupole (Vantage) mass spectrometers. Results: Discovery workflow validated known secretion mechanism proteins in tuberculosis (TB); targeted SRM methods provided quantitative results for specific peptide transitions.