Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.
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ABSTRACT:
SUBMITTER: Addona TA
PROVIDER: S-EPMC2855883 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundIn the Addona et al. paper (Nature Biotechnology 2009), a large-scale multi-site study was performed to quantify Multiple Reaction Monitoring (MRM) measurements of proteins spiked in human plasma. The unlabeled signature peptides derived from the seven target proteins were measured at nine different concentration levels, and their isotopic counterparts were served as the internal standards.Methodology/principal findingsIn this paper, the sources of variation are analyzed by decomposing the variance into parts attributable to specific experimental factors: technical replicates, sites, peptides, transitions within each peptide, and higher-order interaction terms based on carefully built mixed effects models. The factors of peptides and transitions are shown to be major contributors to the variance of the measurements considering heavy (isotopic) peptides alone. For the light ((12)C) peptides alone, in addition to these factors, the factor of study*peptide also contributes significantly to the variance of the measurements. Heterogeneous peptide component models as well as influence analysis identify the outlier peptides in the study, which are then excluded from the analysis. Using a log-log scale transformation and subtracting the heavy/isotopic peptide [internal standard] measurement from the peptide measurements (i.e., taking the logarithm of the peak area ratio in the original scale establishes that), the MRM measurements are overall consistent across laboratories following the same standard operating procedures, and the variance components related to sites, transitions and higher-order interaction terms involving sites have greatly reduced impact. Thus the heavy peptides have been effective in reducing apparent inter-site variability. In addition, the estimates of intercepts and slopes of the calibration curves are calculated for the sub-studies.Conclusions/significanceThe MRM measurements are overall consistent across laboratories following the same standard operating procedures, and heavy peptides can be used as an effective internal standard for reducing apparent inter-site variability. Mixed effects modeling is a valuable tool in mass spectrometry-based proteomics research.
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Project description:Increasing number of studies report the relevance of protein Ser/Thr/Tyr phosphorylation in bacterial physiology, yet the analysis of this type of modification in bacteria still presents a considerable challenge. Unlike in eukaryotes, where tens of thousands of phosphorylation events likely occupy more than two thirds of the proteome, the abundance of protein phosphorylation is much lower in bacteria. Even the state-of-the-art phosphopeptide enrichment protocols fail to remove the high background of abundant unmodified peptides, leading to low signal intensity and undersampling of phosphopeptide precursor ions in consecutive data-dependent MS runs. Consequently, large-scale bacterial phosphoproteomic datasets often suffer from poor reproducibility and a high number of missing values. Here we explore the application of parallel reaction monitoring (PRM) on a Q Exactive mass spectrometer in bacterial phosphoproteome analysis, focusing especially on run-to-run sampling reproducibility. In multiple measurements of identical phosphopeptide-enriched samples, we show that PRM outperforms DDA in terms of detection frequency, reaching almost complete sampling efficiency, compared to 20% in DDA. We observe a similar trend over multiple rounds of (heterogeneous) phosphopeptide-enriched samples and conclude that PRM is a method of choice in bacterial phosphoproteomics projects where reproducible detection and quantification of a relatively small set of phosphopeptides is desired.
2018-06-05 | PXD008211 | Pride
Project description:Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids ([(13)C(6)]Arg or [(13)C(6)]Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.
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Project description:HDL carries a rich protein cargo and examining HDL protein composition promises to improve our understanding of its functions. Conventional mass spectrometry methods can be lengthy and difficult to extend to large populations. In addition, without prior enrichment of the sample, the ability of these methods to detect low abundance proteins is limited. Our objective was to develop a high-throughput approach to examine HDL protein composition applicable to diabetes and cardiovascular disease (CVD).We optimized two multiplexed assays to examine HDL proteins using a quantitative immunoassay (Multi-Analyte Profiling- MAP) and mass spectrometric-based quantitative proteomics (Multiple Reaction Monitoring-MRM). We screened HDL proteins using human xMAP (90 protein panel) and MRM (56 protein panel). We extended the application of these two methods to HDL isolated from a group of participants with diabetes and prior cardiovascular events and a group of non-diabetic controls.We were able to quantitate 69 HDL proteins using MAP and 32 proteins using MRM. For several common proteins, the use of MRM and MAP was highly correlated (p < 0.01). Using MAP, several low abundance proteins implicated in atherosclerosis and inflammation were found on HDL. On the other hand, MRM allowed the examination of several HDL proteins not available by MAP.MAP and MRM offer a sensitive and high-throughput approach to examine changes in HDL proteins in diabetes and CVD. This approach can be used to measure the presented HDL proteins in large clinical studies.
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Project description:Direct quantitation of proteins in complex biological matrices by mass spectrometry remains a challenge. Here, we describe a novel top-down parallel reaction monitoring-mass spectrometry (PRM-MS) assay, enabled by microflow LC-nanospray MS using a silicon microfluidic LC-MS chip. We demonstrated direct analysis of intact proteins such as somatropin in human plasma, achieving sensitivity (0.1-1.0 fmole) and speed (1-5 min) on par with enzyme-linked immunosorbent assay (ELISA).
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Project description:To assess the reproducibility of bone and soft-tissue pelvimetry measurements obtained from dynamic magnetic resonance (MR) imaging studies in primiparous women across multiple centers.All subjects prospectively gave consent for participation in this institutional review board-approved, HIPAA-compliant study. At six clinical sites, standardized dynamic pelvic 1.5-T multiplanar T2-weighted MR imaging was performed in three groups of primiparous women at 6-12 months after birth: Group 1, vaginal delivery with anal sphincter tear (n = 93); group 2, vaginal delivery without anal sphincter tear (n = 79); and group 3, cesarean delivery without labor (n = 26). After standardized central training, blinded readers at separate clinical sites and a blinded expert central reader measured nine bone and 10 soft-tissue pelvimetry parameters. Subsequently, three readers underwent additional standardized training, and reread 20 MR imaging studies. Measurement variability was assessed by using intraclass correlation for agreement between the clinical site and central readers. Acceptable agreement was defined as an intraclass correlation coefficient (ICC) of at least 0.7.There was acceptable agreement (ICC range, 0.71-0.93) for eight of 19 MR imaging parameters at initial readings of 198 subjects. The remaining parameters had an ICC range of 0.13-0.66. Additional training reduced measurement variability: Twelve of 19 parameters had acceptable agreement (ICC range, 0.70-0.92). Correlations were greater for bone (ICC, >or=0.70 in five [initial readings] and eight of nine [rereadings] variables) than for soft-tissue measurements (ICC, >or=0.70 in three [initial readings] of 10 and four [rereadings] of 10 readings, respectively).Despite standardized central training, there is high variability of pelvic MR imaging measurements among readers, particularly for soft-tissue structures. Although slightly improved with additional training, measurement variability adversely affects the utility of many MR imaging measurements for multicenter pelvic floor disorder research.
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Project description:Women are vulnerable to cervical diseases including normal control, HSIL and early stage cervical carcinoma . The parallel reaction monitoring mass spectrometry (PRM-MS) were used to qualitatively and quantitatively analyze the candidate differential proteins identified in our previous studies and to link them with HPV status. The integration of HPV status with candidate serum markers is expected to efficiently narrow down the high risk population and provide sufficient clinical information for early diagnosis and effective therapy.
2021-09-09 | PXD022756 | Pride
Project description:Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.
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Project description:ObjectiveQuantitative magnetic resonance imaging (MRI) evidence of mediobasal hypothalamic (MBH) gliosis positively correlates with body mass index (BMI) in adults. This has neither been well explored in children nor have other brain regions involved in appetitive processing been tested for evidence of gliosis.MethodsMulti-site cross-sectional study in children to test for differences in quantitative T2 signal (measure of gliosis) by region and to assess relationships with age and BMI. Participants underwent brain MRI using the same equipment and protocol to quantify T2 relaxation time in six bilateral regions of interest (ROIs): putamen, caudate, ventral striatum, amygdala, hippocampus and MBH, and three control regions: white matter, motor cortex and dorsal hypothalamus.ResultsThirty-one participants (61% female) were included in a combined sample from the University of Washington (N = 9) and John Hopkins University (N = 22). Mean age was 14 ± 3 years, and BMI z-score was 0.7 ± 1.1 (26% with obesity). No study site-related differences were seen in T2 relaxation time across all nine regions (chi2 (8): 9.46, P = .30). Regional differences in T2 relaxation time were present (P < .001). MBH presented longer T2 relaxation time, suggestive of gliosis, when compared to all regions (P < .001), including an intra-hypothalamic control. Physiological age-related declines in T2 relaxation times were found in grey matter ROIs, but not in the MBH (r = -0.14, P = .46). MBH was the only region with a positive correlation between T2 relaxation time and BMI z-score (r = 0.38, P = .03).ConclusionsIn a multi-site study, pilot data suggest that quantitative MRI detected normal maturation-related brain variation as well as evidence that MBH gliosis is associated with increased adiposity in children.
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Project description:PurposeTo evaluate the repeatability and reproducibility of corneal power measurements obtained by Topcon KR-1W and iTrace, and assess the agreement with measurements obtained by Allegro Topolyzer and IOLMaster.MethodsThe right eyes of 100 normal subjects were prospectively scanned 3 times using all the 4 devices. Another observer performed additional 3 consecutive scans using the Topcon KR-1W and iTrace in the same session. About one week later, the first observer repeated the measurements using the Topcon KR-1W and iTrace. The steep keratometry (Ks), flat keratometry (Kf), mean keratometry (Km), J0 and J45 were analyzed. Repeatability and reproducibility of measurements were evaluated by the within-subject standard deviation (Sw), coefficient of variation (CoV), test-retest repeatability (2.77Sw), and intraclass correlation coefficient (ICC). Agreements between devices were assessed using Bland-Altman analysis and 95% limits of agreement (LoA).ResultsIntraobserver repeatability and interobserver and intersession reproducibility of the Ks, Kf and Km showed a CoV of no more than 0.5%, a 2.77Sw of 0.70 D or less, and an ICC of no less than 0.99. However, J0 and J45 showed poor intraobserver repeatability and interobserver and intersession reproducibility (all ICCs not greater than 0.446). Statistically significant differences existed between Topcon KR-1W and IOLMaster, Topcon KR-1W and iTrace, Topcon KR-1W and Topolyzer, iTrace and Topolyzer, iTrace and IOLMaster for Ks, Kf and Km measurements (all P < 0.05). The mean differences between Topcon KR-1W, iTrace, and the other 2 devices were small. The 95% LoA were approximately 1.0 D to 1.5 D for all measurements.ConclusionsThe Ks, Kf and Km obtained by Topcon KR-1W and iTrace showed excellent intraobserver repeatability and interobserver and intersession reproducibility in normal eyes. The agreement between Topcon KR-1W and Topolyzer, Topcon KR-1W and IOLMaster, iTrace and Topolyzer, iTrace and IOLMaster, Topcon KR-1W and iTrace were not so good, they should not be interchangeable in clinical application. Given that the intraobserver repeatability and interobserver and intersession reproducibility of corneal astigmatism measurements obtained by Topcon KR-1W and iTrace were poor, it should be cautious that Topcon KR-1W and iTrace were applied for the preparation of toric lens implantation.
| S-EPMC4709181 | biostudies-literature