YeastMate: neural network-assisted segmentation of mating and budding events in Saccharomyces cerevisiae.

SUMMARY: Here, we introduce YeastMate, a user-friendly deep learning-based application for automated detection and segmentation of Saccharomyces cerevisiae cells and their mating and budding events in microscopy images. We build upon Mask R-CNN with a custom segmentation head for the subclassification of mother and daughter cells during lifecycle transitions. YeastMate can be used directly as a Python library or through a standalone application with a graphical user interface (GUI) and a Fiji plugin as easy-to-use frontends. AVAILABILITY AND IMPLEMENTATION: The source code for YeastMate is freely available at https://github.com/hoerlteam/YeastMate under the MIT license. We offer installers for our software stack for Windows, macOS and Linux. A detailed user guide is available at https://yeastmate.readthedocs.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Distributed coding of duration in rodent prefrontal cortex during time reproduction.

As we interact with the external world, we judge magnitudes from sensory information. The estimation of magnitudes has been characterized in primates, yet it is largely unexplored in nonprimate species. Here, we use time interval reproduction to study rodent behavior and its neural correlates in the context of magnitude estimation. We show that gerbils display primate-like magnitude estimation characteristics in time reproduction. Most prominently their behavioral responses show a systematic overestimation of small stimuli and an underestimation of large stimuli, often referred to as regression effect. We investigated the underlying neural mechanisms by recording from medial prefrontal cortex and show that the majority of neurons respond either during the measurement or the reproduction of a time interval. Cells that are active during both phases display distinct response patterns. We categorize the neural responses into multiple types and demonstrate that only populations with mixed responses can encode the bias of the regression effect. These results help unveil the organizing neural principles of time reproduction and perhaps magnitude estimation in general.

Cristae-dependent quality control of the mitochondrial genome.

Mitochondrial genomes (mtDNA) encode essential subunits of the mitochondrial respiratory chain. Mutations in mtDNA can cause a shortage in cellular energy supply, which can lead to numerous mitochondrial diseases. How cells secure mtDNA integrity over generations has remained unanswered. Here, we show that the single-celled yeast Saccharomyces cerevisiae can intracellularly distinguish between functional and defective mtDNA and promote generation of daughter cells with increasingly healthy mtDNA content. Purifying selection for functional mtDNA occurs in a continuous mitochondrial network and does not require mitochondrial fission but necessitates stable mitochondrial subdomains that depend on intact cristae morphology. Our findings support a model in which cristae-dependent proximity between mtDNA and the proteins it encodes creates a spatial "sphere of influence," which links a lack of functional fitness to clearance of defective mtDNA.

Regulatory encoding of quantitative variation in spatial activity of a Drosophila enhancer.

Developmental enhancers control the expression of genes prefiguring morphological patterns. The activity of an enhancer varies among cells of a tissue, but collectively, expression levels in individual cells constitute a spatial pattern of gene expression. How the spatial and quantitative regulatory information is encoded in an enhancer sequence is elusive. To link spatial pattern and activity levels of an enhancer, we used systematic mutations of the yellow spot enhancer, active in developing Drosophila wings, and tested their effect in a reporter assay. Moreover, we developed an analytic framework based on the comprehensive quantification of spatial reporter activity. We show that the quantitative enhancer activity results from densely packed regulatory information along the sequence, and that a complex interplay between activators and multiple tiers of repressors carves the spatial pattern. Our results shed light on how an enhancer reads and integrates trans-regulatory landscape information to encode a spatial quantitative pattern.

Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry for Quantitative Amino Acid Analysis.

The role of amino acid analysis in bioanalysis has changed from a qualitative to a quantitative technique. With the discovery of both electrospray ionization and matrix-assisted laser desorption ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry. Accurate measurement of the relative molecular masses of proteins and peptides, peptide mapping, and sequencing by tandem mass spectrometry provide significantly better qualitative information than can be achieved from amino acid analysis. At NIST, amino acid analysis is used to assign concentration values to protein and peptide standard reference materials (SRMs) which, subsequently, will be used in the calibration of a wide variety of protein and peptide assays, such as those used in clinical diagnostics. It is critical that the amino acid analysis method used at NIST for assigning concentration values in SRM deliver the highest accuracy and precision possible. Therefore, we have developed an amino acid analysis method that uses isotope dilution LC-MS/MS-the analytical technique routinely used at NIST to certify analyte concentrations in SRMs for a wide variety of analytes. We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS.

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.

Evaluation of standardization capability of current cardiac troponin I assays by a correlation study: results of an IFCC pilot project.

BACKGROUND: As a part of an International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) project to prepare a commutable reference material for cardiac troponin I (cTnI), a pilot study evaluated current cTnI assays for measurement equivalence and their standardization capability. METHODS: cTnI-positive samples collected from 90 patients with suspected acute myocardial infarction were assessed for method comparison by 16 cTnI commercial assays according to predefined testing protocols. Seven serum pools prepared from these samples were also assessed. RESULTS: Each assay was assessed against median cTnI concentrations measured by 16 cTnI assays using Passing-Bablok regression analysis of 79 patient samples with values above each assay's declared detection limit. We observed a 10-fold difference in cTnI concentrations for lowest to highest measurement results. After mathematical recalibration of assays, the between-assay variation for patient samples reduced on average from 40% to 22% at low cTnI concentration, 37%-20% at medium concentration, and 29%-14% at high concentration. The average reduction for pools was larger at 16%, 13% and 7% for low, medium and high cTnI concentrations, respectively. Overall, assays demonstrated negligible bias after recalibration (y-intercept: -1.4 to 0.3 ng/L); however, a few samples showed substantial positive and/or negative differences for individual cTnI assays. CONCLUSIONS: All of the 16 commercial cTnI assays evaluated in the study demonstrated a significantly higher degree of measurement equivalence after mathematical recalibration, indicating that measurement harmonization or standardization would be effective at reducing inter-assay bias. Pooled sera behaved similarly to individual samples in most assays.

Multiplexed LC-MS/MS assay for urine albumin.

Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urine albumin is vital to clinical diagnosis. Although intermethod differences and analyte heterogeneity have been reported for urine albumin measurements, accuracy assessments of the available methods have been hindered by the lack of a reference system, including reference measurement procedures and reference materials, for this clinical analyte. To address the need for a reference measurement system for urine albumin, we have developed a candidate reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify full-length urine albumin in a targeted mass spectrometric-based approach. The reference measurement procedure incorporates an isotopically labeled ((15)N) full-length recombinant human serum albumin ((15)N-rHSA) material as the internal standard, which permits the absolute quantitation of albumin in urine. A total of 11 peptides with two transitions per peptide were selected from the tryptic digestion of human serum albumin on the basis of retention time reproducibility, peak intensity, and the degree of HSA sequence coverage. In addition to method validation, the generated calibration curves were used to determine the albumin content in pooled human urine samples to access the accuracy of the MS-based urine albumin quantitation method.

QC metrics from CPTAC raw LC-MS/MS data interpreted through multivariate statistics.

Shotgun proteomics experiments integrate a complex sequence of processes, any of which can introduce variability. Quality metrics computed from LC-MS/MS data have relied upon identifying MS/MS scans, but a new mode for the QuaMeter software produces metrics that are independent of identifications. Rather than evaluating each metric independently, we have created a robust multivariate statistical toolkit that accommodates the correlation structure of these metrics and allows for hierarchical relationships among data sets. The framework enables visualization and structural assessment of variability. Study 1 for the Clinical Proteomics Technology Assessment for Cancer (CPTAC), which analyzed three replicates of two common samples at each of two time points among 23 mass spectrometers in nine laboratories, provided the data to demonstrate this framework, and CPTAC Study 5 provided data from complex lysates under Standard Operating Procedures (SOPs) to complement these findings. Identification-independent quality metrics enabled the differentiation of sites and run-times through robust principal components analysis and subsequent factor analysis. Dissimilarity metrics revealed outliers in performance, and a nested ANOVA model revealed the extent to which all metrics or individual metrics were impacted by mass spectrometer and run time. Study 5 data revealed that even when SOPs have been applied, instrument-dependent variability remains prominent, although it may be reduced, while within-site variability is reduced significantly. Finally, identification-independent quality metrics were shown to be predictive of identification sensitivity in these data sets. QuaMeter and the associated multivariate framework are available from http://fenchurch.mc.vanderbilt.edu and http://homepages.uc.edu/~wang2x7/ , respectively.

A reference system for urinary albumin: current status.

BACKGROUND: Increased urinary excretion of albumin reflects kidney damage and is a recognized risk factor for progression of renal and cardiovascular disease. Considerable inter-method differences have been reported for both albumin and creatinine measurement results, and therefore the albumin-to-creatinine ratio. Measurement accuracy is unknown and there are no independent reference measurement procedures for albumin and no reference materials for either measurand in urine. METHODS: The National Kidney Disease Education Program (NKDEP) Laboratory Working Group and the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) have initiated joint projects to facilitate standardization of urinary albumin and creatinine measurement. RESULTS: A candidate LC-MS/MS reference measurement procedure for urinary albumin and candidate reference materials for urinary albumin and creatinine has been developed. The status of validations of these reference system components is reported. CONCLUSIONS: The development of certified reference materials and reference measurement procedures for urinary albumin will enable standardization of this important measurand.

Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry.

Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID-MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of (15)N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC-MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of (15)N-rCRP as an internal standard in ID-MS. Standardization of CRP assays should help validate the relationship between CRP and human health.

A proteomics performance standard to support measurement quality in proteomics.

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods.

Isotope dilution liquid chromatography-tandem mass spectrometry for quantitative amino acid analysis.

The role of amino acid analysis in bioanalysis has changed from a qualitative to a quantitative technique. With the discovery of both electrospray ionization and matrix-assisted laser desorption ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry. Accurate measurement of the relative molecular masses of proteins and peptides, peptide mapping, and sequencing by tandem mass spectrometry provide significantly better qualitative information than can be achieved from amino acid analysis. At NIST, amino acid analysis is used to assign concentration values to protein and peptide standard reference materials (SRMs) which, subsequently, will be used in the calibration of a wide variety of protein and peptide assays, such as those used in clinical diagnostics. It is critical that the amino acid analysis method used at NIST for SRM measurement deliver the highest accuracy and precision possible. Therefore, we have developed an amino acid analysis method that uses isotope dilution LC-MS/MS - the analytical technique routinely used at NIST to certify analyte concentrations in SRMs for a wide variety of analytes. Amino acid analysis by isotope dilution LC-MS/MS was first used to measure the concentration of bovine serum albumin in NIST SRM 927d ("bovine serum albumin, 7% solution"). We have recently refined our isotope dilution LC-MS/MS amino acid analysis method to certify the concentration of 17 amino acids in NIST SRM 2389a ("amino acids in 0.1 mol/L hydrochloric acid"). We present here our most recent method for the quantification of amino acids using isotope dilution LC-MS/MS.

A quantitative LC-MS/MS method for comparative analysis of capture-antibody affinity toward protein antigens.

A mass spectrometry-based antibody selection procedure was developed to evaluate optimal 'capture' monoclonal antibodies that can be used in a variety of analytical measurement applications. The isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) methodology is based on the use of multiple-reaction monitoring of tryptic peptide fragments derived from protein antigens. A panel of monoclonal antibodies (mAb) was evaluated based on a quantitative determination of relative binding affinity to human cardiac troponin I following immunoprecipitation. Dissociation constants (K(d)) were determined for 'bound mAb-antigen' vs. 'unbound antigen' using non-linear regression analysis. Relative quantification of both antigen and antibody was based on the use of stable isotope-labeled synthetic peptides as internal standards. Optimal 'capture' mAbs were determined through evaluation of relative K(d) constants of all monitored peptide transitions. A panel of six pre-screened candidate capture mAbs was concluded to consist of two subsets of mAbs, each with statistically equivalent K(d) constants as determined using NIST Standard Reference Material (SRM) 2921 - Human Cardiac Troponin Complex. This ID LC-MS/MS method is shown to be capable of quantitatively differentiating mAbs based on relative binding affinities. Selection of optimal capture mAbs can be applied toward a number of analytical applications which require metrological traceability and unbiased quantification.

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.

An immunoprecipitation coupled with fluorescent Western blot analysis for the characterization of a model secondary serum cardiac troponin I reference material.

BACKGROUND: Cardiac troponin I (cTnI) is considered the 'gold standard' cardiac biomarker. However, the result comparability of commercial cTnI immunoassays is still lacking despite the availability of NIST Standard Reference Material, SRM 2921 (human cardiac troponin). To facilitate the standardization of the cTnI immunoassays, a secondary reference material consisting of a panel of three cTnI-positive human serum pools is proposed by the IFCC Working Group on Standardization of Troponin I. The objective of this study is to develop measurement procedures for the characterization of the future secondary reference material using a pooled cTnI-positive serum sample as a development model. METHODS: We used magnetic beads coupled with 6 different anti-cTnI monoclonal antibodies that bind specifically to different amino acid sequence regions of the cTnI molecule to immunoprecipitate cTnI proteins from the pooled cTnI-positive serum sample followed by sensitive detection using a fluorescent Western blot. RESULTS: The degradation of cTnI in the pooled sample was detected and the concentration of cTnI was determined. CONCLUSION: We demonstrated the utility of this measurement procedure in support of the development of the proposed secondary cTnI-positive, serum-based reference material.

Development of a candidate secondary reference procedure (immunoassay based measurement procedure of higher metrological order) for cardiac troponin I: I. Antibody characterization and preliminary validation.

In this study, the first steps in the development of a secondary reference measurement procedure (RMP) 'higher metrological order measurement procedure' to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 µg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.

Design considerations for proteomic reference materials.

In order to improve the repeatability, comparability, and accuracy of MS-based proteomic measurements, there has been considerable international effort to develop appropriate reference materials. Although the majority of reference materials are developed to support measurement quality of routine assays, the development of reference materials for a diverse and changing research field such as proteomics represents unique challenges. In order to define common measurement components and common features of typical proteomic samples, the metrology underpinning proteomics must be considered due to the diversity and changing nature of the field. Reference materials can then be designed around common aspects in order to produce reference materials with the broadest applicability. Reference materials are needed to support both qualitative and quantitative proteomic measurements, involving different design considerations. Consensus and validated statistical approaches to describe the confidence in qualitative measurement, such as protein identification, needs to be established. Common sources of measurement bias also need to be considered in proteomic reference material design.

Standardisation of cardiac troponin I measurement: past and present.

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.