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...
The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and
A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.
BACKGROUND: Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. CONTENT:The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the error-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences have been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no reference materials for either analyte in urine. The recommended reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethnicity) nor the continuous increase in risk related to albumin excretion. DISCUSSION:Clinical needs have been identified for standardization of (a) urine collection methods, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.
Optimal performance of LC-MS/MS platforms is critical toAccess to proteomics performance standards is essential for several reasons. First, to generate the highest quality data possible, proteomics laboratories routinely benchmark and perform quality control (QC) 1 monitoring of the performance of their instrumentation using standards. Second, appropriate standards greatly facilitate the development of improvements in technologies by providing a timeless standard with which to evaluate new protocols or instruments that claim to improve performance. For example, it is common practice for an individual laboratory considering purchase of a new instrument to require the vendor to run "demo" samples so that data from the new instrument can be compared head to head with existing instruments in the laboratory. Third, large scale proFrom the
BACKGROUND For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope–labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials—in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry—is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.
The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450 cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed in Escherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450 cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.Multiprotein redox enzyme systems such as methane monooxygenase, cytochrome P450s, and diooxygenases of similar molecular architecture are being investigated as biocatalysts for conversion of organic substrates with no functional groups into oxygen-bearing compounds with high regio-or stereo-selectivity. Maximizing the catalytic efficiency of such systems requires knowledge of the pathways of electron transfer and of the surface regions and amino acid residues involved in the interaction of the redox partner subunits.Cytochrome P450 cam (CYP101) has been intensively investigated for over 20 years as a model P450 system (1). This soluble P450 (from Pseudomonas putida) consists of three subunits: putidaredoxin reductase (PdR, 1 M r Ϸ 43,500), putidaredoxin (Pdx, M r Ϸ 11,600), and cytochrome P450 cam hydroxylase (CYP101, M r Ϸ 45,000). The genes, camA (PdR), camB (Pdx), and camC (CYP101) from the cam operon have been cloned and sequenced, and the protein subunits were expressed in individual clones (2-5). Structural information is available for two of the three subunits of the CYP101 system. CYP101 has been crystallized in a number of states, and the structure is well defined (6 -8). Structural information on Pdx comes from solution 1 H NMR studies by Pochapsky and co-workers (9 -11), and they have proposed a model.Electron transfer in this system proceeds from NADH via the flavin group of PdR to the 2Fe-2S center of Pdx and then to the heme iron of CYP101 which accepts one electron at a time from Pdx. Because the details of the electron transfer pathway from one subunit to the next are missing, it is not known exactly how the subunits bind for the transfer of electrons. Ionic strength is well known to have an effect on binding and electron transfer suggesting that salt bridges are important in these interactions (12, 13). The role of some amino acid residues, specifically Trp-106 on Pdx and Arg-112 on CYP101, is known to be important for binding and electron transfer (14 -18). Residues involved in the PdR-Pdx interaction are not necessarily the same as tho...
3,3',5-Triiodothyronine (T3) is an important diagnostic marker for thyroid function. A reference measurement procedure (RMP) for total T3 in serum involving isotope dilution coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The method uses solid-phase extraction with mixed-mode retention mechanisms of reversed phase and ion exchange prior to reversed-phase LC/MS/MS. In addition to a labeled T3 internal standard (T3-13C9), labeled thyroxine (T4-d5) is also added to serum samples in order to monitor the degradation of T4 to T3. The accuracy of the measurement was evaluated by a recovery study for added T3 and was supported by a comparison study with the other RMP. The recovery of the added T3 ranged from 98.9% to 99.4%. The results of this method and the other RMP agreed to within 1%. Samples of frozen serum pools were prepared and measured in three separate sets. Excellent reproducibility was obtained with within-set coefficients of variation (CVs) ranging from 0.8% to 1.6% and between-set CVs ranging from 1.9% to 2.6%. Excellent linearity was also obtained with correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) ranging from 0.9995 to 0.9996. The detection limit at a signal-to-noise ratio of approximately 3 was 1 pg of T3. The T4 degradation during sample preparation was minimized to a small percentage (no more than 3% of the T3 values) by use of antioxidants (ascorbic acid, dithiothreitol, citric acid) and can be accounted for in the T3 measurement process. This well-characterized LC/MS/MS method for total serum T3, which demonstrates good accuracy and precision, low susceptibility to interferences, accountability of the conversion of T4 to T3, and comparability with the other RMP, qualifies as a reference measurement procedure and can be used to provide an accuracy base to which routine methods for T3 can be compared.
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