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PubMed2026

Reframing Proteomics Measurement: Super Mass Spectrometry Framework and the Role of Delayed Electrospray Ionization Technique.

Dynamic range, repeatability, and reproducibility remain the central limitations of data-independent acquisition (DIA) proteomics. Current workflows emphasize protein group identification counts and throughput, but these metrics mask the fundamental measurement challenge: generating a repeatable, reproducible, high-fidelity, and relatively complete digital representation of complex proteomes. In particular, plasma proteomics spans more than 10 orders of magnitude in protein abundance, far exceeding the capacity and dynamic range of any single mass spectrometer. Incremental advances have not closed this gap. In this Perspectives article, I introduce the Super Mass Spectrometry framework and then highlight the Delayed Electrospray Ionization (Delayed-ESI) technique, as a practical approach to address these limitations. By producing compositionally identical but temporally staggered ion beams, the Delayed-ESI technique enables deterministic remeasurement of the same analyte profile, supporting various novel strategies to improve analytical figures of merit. While recent implementations of the Delayed-ESI technique have emphasized throughput, I argue that the broader value of the Delayed-ESI technique lies in extending dynamic range and improving repeatability and reproducibility─objectives that should take precedence if proteomics is to evolve into a robust measurement science capable of supporting population-scale proteomics studies.

Proteomics↗
PubMed1991

A statistical procedure for the estimation of accuracy parameters in interlaboratory studies.

Interlaboratory studies are conducted to estimate the accuracy of methods of laboratory measurements. The standard parameters used to describe this accuracy are the repeatability and the reproducibility. Usually variance components models are used to estimate these parameters. If the model assumptions are violated the resulting estimates for reproducibility and repeatability may, however, be biased. A new method of residual analysis in variance components models--developed by the author--may be used to detect violations of the model assumptions. If the residual analysis indicates that the model assumptions are violated, a simple robust method--which makes fewer assumptions--may be used for the estimation of accuracy parameters. The application of this residual analysis is demonstrated using data of an interlaboratory study. Graphical methods play an important role in the evaluation of the residuals. The analysis of the residuals uses methods similar to those used for the analysis of Studentized residuals in the linear model. The estimates obtained by the variance components model and the simple robust method are compared. The results of the residual analysis may be used to decide which of the two estimates can be considered more appropriate. The necessity of residual analysis in the analysis of interlaboratory studies by variance components models is pointed out. Potential hazards inherent to residual analysis in variance components models are discussed. Conclusions for the analysis of interlaboratory studies are drawn.

Bias↗
PubMed1995

The reproducibility of reported height and body weight in repeated questionnaire surveys.

Reproducibility of reported height and weight were studied via repeated questionnaire surveys distributed to subjects within a year. Effects of the factors which might influence the reproducibility of these values were investigated. Although only about half of the subjects reported the same height and 30% the same weight on the second questionnaire, no change was found in average height and weight after a one year interval for both male and female subjects. The reproducibility decreased with age for both height and weight. Smokers tended to have lower reproducibility for height than non-smokers. The shorter subjects had lower height reproducibility than their taller counterparts, while heavier subjects had lower weight reproducibility than light subjects. It was observed that the lower the weight reproducibility, the lower the height reproducibility and vice versa. Reported height or weight figures ending in zero or five were more frequent than the expected 10% with low reproducibility.

Adult↗
PubMed2026

Repeated drought induces a reproducible DNA methylation response associated with gene expression in Quercus lobata.

UNLABELLED: Long-lived trees must continually adjust to environmental change and face sustained climatic shifts over their lifetimes. One increasingly important challenge is the rising frequency of drought caused by climate change. Environmentally responsive DNA methylation is widespread in plants, but whether it contributes to gene expression during environmental stress remains unclear, particularly in long-lived trees. Here, we integrated long read methylomes and transcriptomes from valley oak ( Quercus lobata ) seedlings exposed to repeated drought and well-watered treatments. Repeated drought induced a reproducible DNA methylation response that repeatedly targeted the same genomic regions despite turnover of individual methylated sites. These repeatedly targeted regions were transposable elements (TEs) located near genes. Genes adjacent to CHH-methylated TEs were enriched for core drought-response pathways, including abscisic acid signaling, osmotic adjustment and cell-wall remodeling, and remained transcriptionally activated under drought. However, higher CHH methylation levels were associated with progressively smaller transcriptional responses, suggesting that environmentally responsive DNA methylation influences how strongly drought- response genes are activated rather than simply switching them on or off. At the same time, greater CHH methylation was associated with continued repression of nearby TEs, suggesting that this response may simultaneously regulate gene activity while maintaining genome stability. Together, these findings identify a reproducible genome- regulatory response associated with repeated environmental stress in a long-lived tree. By repeatedly targeting the same genomic regions despite turnover of individual sites, this response provides a framework for how long-lived trees repeatedly adjust gene expression while maintaining genome stability during environmental change. SIGNIFICANCE STATEMENT: Plants cannot escape environmental change, and trees must repeatedly respond to stresses, such as drought, over lifetimes spanning decades to centuries. Yet little is known about the molecular mechanisms that make this remarkable resilience possible. Using a widespread California oak, we show that repeated drought repeatedly induced the same DNA methylation pattern in the same parts of the genome, even though the differentially methylated individual sites changed between drought events. This pattern was linked to how strongly drought-response genes were activated, suggesting that trees repeatedly deploy the same molecular program to respond to environmental stress. Our findings provide a new framework for understanding how long-lived organisms repeatedly adjust to changing climates.

Journal Article↗
PubMedDate not supplied

Enzymatic-ultraviolet determination of glucose and fructose in wine: collaborative study.

This collaborative study on the determination of glucose and fructose in wine was performed by 18 laboratories on 4 matched pairs of commercial wine. The method uses the enzymes hexokinase, glucose-6-phosphate dehydrogenase, and phosphoglucose isomerase and the coenzyme nicotinamide-adenine dinucleotide phosphate. Both glucose and fructose can be determined in the same sample without separation. The method is simple but care is necessary to ensure precise transfer of small volumes. Repeatability and reproducibility standard deviations for glucose ranged from 2.6 to 14.6 mg/L and 4.7 to 16.5 mg/L, respectively. Repeatability and reproducibility values for fructose ranged from 2.4 to 16.1 mg/L and 6.0 to 21.3 mg/L, respectively. The method has been adopted official first action.

Fructose↗
PubMed1982

Collaborative evaluation of rolling ball viscometer for measuring somatic cells in abnormal milk.

The Ruakura rolling ball viscometer was evaluated in 3 laboratories along with currently approved instrumental methods for measuring somatic cells in milk and the Wisconsin mastitis test. Replacement of the Teepol reagent with Wisconsin mastitis test reagent in the rolling ball viscometer was also evaluated. Both repeatability and reproducibility were satisfactory for all methods evaluated. The instrumental methods each gave higher readings than the other 3 methods. Use of Wisconsin mastitis test reagent in the rolling ball viscometer improved both repeatability and reproducibility. Additional work on standardization is suggested to match rolling ball viscometer readings with those of the instrumental methods.

Animals↗
PubMedDate not supplied

Liquid chromatographic determination of penicillin V potassium in tablets: collaborative study.

A liquid chromatographic method for the determination of penicillin V potassium in tablets was collaboratively studied by 7 laboratories. The method uses an octadecyl silane reverse-phase column, a mobile phase of acetonitrile-methanol-0.01 M potassium phosphate monobasic (21 + 4 + 75 v/v/v), photometric detection at 225 nm, and sulfadimethoxine as an internal standard. Each collaborator received 6 samples: powdered composites of 2 commercial tablet preparations and 1 synthetic tablet powder mixture, each with blind duplicates. The mean repeatability and reproducibility relative standard deviations for commercial samples were, respectively, 1.10 and 1.46% (250 mg dosage), and 0.84 and 2.82% (500 mg dosage). The average standard recovery from the synthetic formulation was 99.1%, with repeatability and reproducibility relative standard deviations of 1.30 and 3.66%, respectively. The method has been adopted official first action.

Chromatography, Liquid↗
PubMed1979

Collaborative study of a GLC method for vitamin E.

The official GLC method of the Association of Official Analytical Chemists (AOAC) for determining vitamin E was modified and collaboratively studied for the National Formulary (NF). The internal standard hexadecyl hexadecanoate (cetyl palmitate) was substituted for the dotriacontane used in the AOAC method, and some other minor changes were made. Eleven samples, representing all types of NF formulations and NF bulk materials, were analyzed by 11 laboratories. The coefficients of variation of the reproducibility and repeatability were 4.5 and 2.4%, respectively, for all laboratories and samples. The values were 3.4 and 1.6%, respectively, when the one laboratory statistically determined to be an outlier was excluded. The coefficients of variation of reproducibility and repeatability for alpha-tocopheryl acid succinate were 2.1 and 1.5%, respectively. All of these values lie within the 5% limit required by the NF.

Analysis of Variance↗
PubMedDate not supplied

Determination of ethanol in wine by titrimetric and spectrophotometric dichromate methods: collaborative study.

A dichromate-spectrophotometric method for the determination of ethanol in wine was compared in a collaborative, matched pair study with the AOAC dichromate-titrimetric method, 11.008-11.011. Both methods require distillation of the sample into dichromate. The titrimetric method measures ethanol by titrating the excess dichromate with ferrous ammonium sulfate after conversion of ethanol to acetic acid; the spectrophotometric method directly measures the reduced dichromate formed after oxidation. In addition to comparing the 2 methods, the collaborative study also compared the use of 2 types of assemblies for obtaining the ethanol distillate: the Scott-type, which is used in 11.008-11.011, and the electric Kirk-type. Results of the collaborative study indicated that the repeatability and reproducibility of the official titrimetric method were generally far superior to those of the spectrophotometric method; therefore, adoption of the spectrophotometric method is not recommended. Comparison of titrimetric method results obtained using the 2 types of stills indicated that repeatability and reproducibility were somewhat better when Scott apparatus was used, but measurements using Kirk-type compared well in the range of ethanol concentrations found in table and fortified wines. The Kirk-type distillation apparatus has been adopted official first action as an alternative to Scott apparatus in the dichromate oxidation method for ethanol in wine, 11.008-11.011.

Chromates↗
PubMed1986

Evaluation of an ISO draft proposal for sampling and analysis of chlorinated hydrocarbon solvent vapors in workplace atmospheres.

The ISO draft proposal for sampling and analysis of chlorinated hydrocarbon solvent vapors in workplace atmospheres (ISO/TC 146/SC 2/WG4 N41) has been evaluated by means of a round-robin test. For this purpose, the repeatability and reproducibility for the determination of tetrachloromethane, trichloromethane, 1,1,1-trichloroethane, trichloroethene were determined at three concentration levels. Furthermore, parameters such as breakthrough and loss on storage were determined. Samples were taken by 11 participants from six countries. After statistical evaluation of the results, it appeared that improvements were necessary for the determination of tetrachloromethane and trichloromethane. For the other three compounds, repeatability and reproducibility were sufficient. Possible error sources and possibilities for improvements are discussed.

Air Pollutants, Occupational↗
PubMed1989

Clinical usefulness of ultrashort-lived iridium-191m from a carbon-based generator system for the evaluation of the left ventricular function.

Ultrashort-lived 191mIr (4.96 sec; 63-74 and 129 keV photons) is potentially advantageous for first-pass radionuclide angiocardiography, offering the opportunity to perform repeat studies with very low absorbed radiation dose to the patient. Left ventricular (LV) first-pass studies were performed in 72 patients with 191mIr from a new bedside 1.3 Ci (48.1 GBq) 191Os/191mIr generator system using an activated carbon support that offers high 191mIr yields (15-18%) and consistent low 191Os breakthrough (2-4 x 10(-4)%/bolus). Using a single crystal digital gamma camera, uncorrected end-diastolic counts in the left ventricular representative cycle ranged from 10 up to 30 k counts. The reproducibility of repeated LV ejection fraction (LVEF) determination at 2-min intervals in 50 patients was r = 0.97, mean diff. = 2.08 +/- 1.55 EF units. Comparison between 191mIr (80-120 mCi; 2,960-4,400 MBq) and 99mTc (20-25 mCi; 750-925 MBq) LV count rates indicates a 3 wk useful shelf life of this new generator system for cardiac studies. Iridium-191m determined LVEF correlated closely with 99mTc determined LVEF in 32 patients (r = 0.96, mean diff. = 1.87 +/- 1.23 EF units). Parametric images for LV wall motion analysis were comparable with both isotopes. We conclude that rapid, repeat, and reproducible high count rate first-pass left ventricular studies can be obtained with 191mIr from this new 191Os/191mIr generator system using a single crystal digital gamma camera.

Adult↗
PubMed1994

Ca2+ mobilization by caffeine in single smooth muscle cells of the rat tail artery.

The fluorescent dye fura-2 was used to study the effects of caffeine on cytosolic free Ca2+ level ([Ca2+]i) in freshly isolated single cells from the rat tail artery. Caffeine caused a concentration-dependent transient increase in [Ca2+]i and shortening of the cell. At higher concentrations (> 2 mM), a tonic increase in [Ca2+]i was also observed. The caffeine-induced changes in [Ca2+]i were reproducible with repeated challenges, even though the cells had contracted due to previous exposure to caffeine. Removal of extracellular Ca2+ reduced the resting [Ca2+]i to about half and abolished the tonic Ca2+ increase to caffeine. The transient component was not significantly affected to the first caffeine challenge after Ca2+ removal, but was abolished to the second challenge. Ryanodine (10 microM) significantly inhibited the responses to caffeine while nifedipine and TMB-8-(8-(diethylamino)octyl ester of 3,4,5-trimethoxybenzoic acid) were not effective. Thapsigargin (10-100 microM) induced a sustained increase in [Ca2+]i to 67 nM. The response of caffeine was not affected by thapsigargin. Pretreatment of the cells with noradrenaline (10 microM) abolished subsequent response to caffeine. These results show that Ca2+ responses to caffeine in single cells from the rat tail artery are reproducible with repeated caffeine challenge. Therefore, single cells can be used for comparison studies of the effects of pharmacological agents.

Animals↗
PubMed1994

Fluorescent-based typing of the two short tandem repeat loci HUMTH01 and HUMACTBP2: reproducibility of size measurements and genetic variation in the Swedish population.

The aim of this study was to investigate the reproducibility of genetic typing of two tetrameric short tandem repeat (STR) loci and the extent of genetic variation in the Swedish population. An automated, fluorescent-based Applied Biosystems 373A sequencer was used for typing of the HUMTH01 and HUMACTBP2 loci (also named SE33). The former locus has seven alleles in the size range of 154-174 bp, while the latter is a complex locus with more than 32 alleles in the range of 227-316 bp. Using different fluorescent dyes, polymerase chain reaction (PCR) products from the two STR loci were sized in one lane using an internal size standard. In order to compare within- and between-gel reproducibility of fragment size estimates, a control sample was typed three times on each of 20 gels. Within the gel, the standard deviation (SD) of fragment size variability was less than 0.1 bp for four fragment sizes between 158-291 bp. Standard deviations between gels were slightly higher for the two shorter fragment sizes (HUMTH01), while the larger fragments varied between 0.3 and 0.4 bp (HUMACTBP2). The amount of genetic variation was investigated in samples from three Swedish cities (n = 301). Seven alleles were found at HUMTH01 and the observed heterozygosity was 0.77. At the HUMACTBP2 locus more than thirty alleles were found and the observed heterozygosity was 0.96. The observed genotype frequencies at HUMTH01 and HUMACTBP2 did not deviate significantly from Hardy-Weinberg expectations. No indication of a significant excess of homozygotes was found at any of the loci. We conclude that both HUMTH01 and HUMACTBP2 can be reliably typed using the method described. However, the latter locus requires an allelic ladder to be run on each gel.

Alleles↗
PubMedDate not supplied

Liquid chromatographic method for determination of aflatoxins B1, B2, G1, and G2 in corn and peanut products: collaborative study.

A collaborative study of a liquid chromatographic method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, Canada, South Africa, and Switzerland. Twenty-one artificially contaminated raw peanuts, peanut butter, and corn samples containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The test portion was extracted with methanol-0.1N HCl (4 + 1), filtered, defatted with hexane, and then partitioned with methylene chloride. The concentrated extract was passed through a silica gel column. Aflatoxins B1 and G1 were derivatized with trifluoroacetic acid, and the individual aflatoxins were determined by reverse-phase liquid chromatography with fluorescence detection. Statistical analysis of the data was performed to determine or confirm outliers, and to compute repeatability and reproducibility of the method. For corn, relative standard deviations for repeatability (RSDr) for aflatoxin B1 ranged from 27.2 to 8.3% for contamination levels from 5 through 50 ng/g. For raw peanuts and peanut butter, RSDr values for aflatoxin B1 were 35.0 to 41.2% and 11.2 to 19.1%, respectively, for contamination levels from 5 through 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. Relative standard deviations for reproducibility (RSDr) for aflatoxin B1 ranged from 15.8 to 38.4%, 24.4 to 33.4%, and 43.9 to 54.0% for corn, peanut butter, and raw peanuts, respectively. The method has been adopted official first action for the determination of aflatoxins B1, B2, G1, and G2 in peanut butter and corn at concentrations greater than or equal to 13 ng total aflatoxins/g.

Aflatoxin B1↗
PubMed1978

Reproducibility and comparative analysis of repeated intravenous and oral glucose tolerance tests.

We have developed a methodology for measuring the reproducibility of the oral glucose tolerance test (OGTT) and the intravenous glucose tolerance test (IVGTT) in normal subjects and in offspring of conjugal diabetic parents. Both groups of subjects revealed more striking correlations of several parameters of blood glucose and insulin secretion between two IVGTTs than between two OGTTs. Employing arbitrary criteria, we calculated a "reproducibility index" as a quantitative measure of blood glucose variability in each subject. No significant difference was found in the reproducibility of OGTT versus IVGTT, nor in normals versus the offspring. Only about 50 per cent of the tests in normals and in the offspring could be considered to be "reproducible." The offspring revealed greater correlations of several parameters, particularly insulin secretion, between the two IVGTTs and between the two OGTTs as compared with the normal group. However, the blood glucose variations tended to be considerably greater in the offspring from one to the other test.

Administration, Oral↗
PubMed1987

Gastric acid secretory capacity falls after repeated within-day pentagastrin testing in fed subjects.

Between-day pentagastrin testing yields highly reproducible stimulated gastric acid output values, but little is known of the reproducibility of repeated within-day pentagastrin tests. We have performed three pentagastrin tests within the 1 day in nine healthy subjects. Within-day tests were 6 hours apart; the first followed an overnight fast and the second and third were both 4 hours after a substantial meal. A further test was performed the following morning, again after an overnight fast, which allowed comparison of within-day and between-day testing. In the second and third within-day tests there was a marked decrease of stimulated gastric acid output, with both maximal and peak acid output decreased to approximately half of the value of the first test (P less than 0.01). By contrast there were no significant differences in the acid output values obtained in between-day tests (both following an overnight fast). Possible mechanisms for the decreased output on repeated within-day testing include alterations in the sensitivity of the gastrin receptor, or some neurohumoral influence secondary to the preceding meal. Future studies of the duration of action of drugs affecting acid secretion may need to take account of these findings.

Adult↗
PubMed1994

Characterization of mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat.

1. Single-unit activity was recorded from S1 sacral dorsal root afferent fibers in the anesthetized rat. A total of 364 afferent fibers were identified by electrical stimulation of the pelvic nerve and subsequently tested for response to colorectal distension (CRD) and urinary bladder distension (UBD). Sixty-seven percent (n = 244) of the fibers were unmyelinated C-fibers and 33% (n = 120) were thinly myelinated A delta-fibers. 2. In three initial experiments, 35 fibers were identified by pelvic nerve stimulation and tested for response only to CRD; none of these fibers responded to CRD. In 20 subsequent experiments, 329 pelvic nerve afferent fibers were tested for response to CRD and UBD. Thirty-four percent (n = 112) of the 329 fibers were unresponsive to noxious CRD (80 mmHg) or to UBD (slow filling < or = 100 mmHg), 44% (n = 146) responded to UBD, 16% (n = 53) responded to CRD, and 6% (n = 18) responded to mechanical stimulation of the anal mucosa. 3. Of the total of 53 pelvic nerve afferent fibers that responded to CRD, 43 (81%) were C-fibers (mean: 1.5 m/s) and 10 (19%) were A delta-fibers (mean: 4.7 m/s). Fifteen of the CRD-sensitive fibers had no resting activity, whereas 38 fibers exhibited some resting activity (mean: 2.6 imp/s). 4. Reproducibility of responses to repeated CRD (80 mmHg, 30s, 10 trials at 4-min intervals) was tested in 17 fibers. In 16, responses to repeated distension were reproducible without evidence of facilitation or inhibition of subsequent responses. One fiber gave greater responses during the 9th and 10th trials. 5. Responses to graded CRD were studied in 44 fibers. All fibers exhibited monotonic, increasing stimulus-response functions < or = 80 mmHg of distension. Thresholds for response of the 44 fibers were determined after extrapolation of the least-squares linear-regression line to the ordinate and varied between 0 and 40 mmHg. Two populations of pelvic nerve afferent fibers in the colon were apparent: low threshold (LT) afferent fibers had a mean threshold of 2.9 mmHg (range: 0-10 mmHg; n = 34) and high threshold (HT) afferent fibers had a mean threshold of 32.6 mmHg (range: 28.0-40.0 mmHg; n = 10). 6. Chemosensitivity to bradykinin (BK) was tested in nine LT fibers. Seven fibers responded to BK (0.1 to 100 micrograms/kg ia) and two fibers did not respond up to 100 micrograms/kg of BK. Responses to BK tested in three fibers were dose dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Anal Canal↗