David M. Bunk

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.

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography−Tandem Mass Spectrometry

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 reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Current Issues in Measurement and Reporting of Urinary Albumin Excretion

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.

Performance Metrics for Liquid Chromatography-Tandem Mass Spectrometry Systems in Proteomics Analyses

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.

Roadmap for Harmonization of Clinical Laboratory Measurement Procedures

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments.

Standardisation of cardiac troponin I measurement: past and present

&NA; The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut‐off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between‐method variation was approximately 2‐ to 5‐fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum‐based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long‐term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG‐TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.

Toward Système International d’Unité-traceable protein quantification: From amino acids to proteins

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.

Probing the interactions of putidaredoxin with redox partners in camphor P450 5-monooxygenase by mutagenesis of surface residues.

The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed inEscherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450cam 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.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

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.