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.

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.

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.

Regulatory encoding of quantitative variation in spatial activity of a 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.

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ionization in the early 1990s, the use of amino acid analysis for qualitative analysis of proteins and peptides has been replaced by mass spectrometry.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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Certification of NIST standard reference material 2389a, amino acids in 0.1 mol/L HCl--quantification by ID LC-MS/MS.

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a-amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer.

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.

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.

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.

Reference measurement procedure development for C-reactive protein in human serum.

This paper describes the development of a reference measurement procedure to quantify human C-reactive protein (CRP) in serum using affinity techniques prior to tryptic digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the certification of reference materials in clinically relevant ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and LC-MS/MS analysis of CRP tryptic peptides.