共查询到20条相似文献,搜索用时 31 毫秒
1.
Laura M. Raffield Hong Dang Katherine A. Pratte Sean Jacobson Lucas A. Gillenwater Elizabeth Ampleford Igor Barjaktarevic Patricia Basta Clary B. Clish Alejandro P. Comellas Elaine Cornell Jeffrey L. Curtis Claire Doerschuk Peter Durda Claire Emson Christine M. Freeman Xiuqing Guo Annette T. Hastie Gregory A. Hawkins Julio Herrera W. Craig Johnson Wassim W. Labaki Yongmei Liu Brett Masters Michael Miller Victor E. Ortega George Papanicolaou Stephen Peters Kent D. Taylor Stephen S. Rich Jerome I. Rotter Paul Auer Alex P. Reiner Russell P. Tracy Debby Ngo Robert E. Gerszten Wanda K. O'Neal Russell P. Bowler 《Proteomics》2020,20(12)
Novel proteomics platforms, such as the aptamer‐based SOMAscan platform, can quantify large numbers of proteins efficiently and cost‐effectively and are rapidly growing in popularity. However, comparisons to conventional immunoassays remain underexplored, leaving investigators unsure when cross‐assay comparisons are appropriate. The correlation of results from immunoassays with relative protein quantification is explored by SOMAscan. For 63 proteins assessed in two chronic obstructive pulmonary disease (COPD) cohorts, subpopulations and intermediate outcome measures in COPD Study (SPIROMICS), and COPDGene, using myriad rules based medicine multiplex immunoassays and SOMAscan, Spearman correlation coefficients range from ?0.13 to 0.97, with a median correlation coefficient of ≈0.5 and consistent results across cohorts. A similar range is observed for immunoassays in the population‐based Multi‐Ethnic Study of Atherosclerosis and for other assays in COPDGene and SPIROMICS. Comparisons of relative quantification from the antibody‐based Olink platform and SOMAscan in a small cohort of myocardial infarction patients also show a wide correlation range. Finally, cis pQTL data, mass spectrometry aptamer confirmation, and other publicly available data are integrated to assess relationships with observed correlations. Correlation between proteomics assays shows a wide range and should be carefully considered when comparing and meta‐analyzing proteomics data across assays and studies. 相似文献
2.
Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides. 相似文献
3.
Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry (HILIC–ESI–MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC–pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites. 相似文献
4.
What is targeted proteomics? A concise revision of targeted acquisition and targeted data analysis in mass spectrometry
下载免费PDF全文
![点击此处可从《Proteomics》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Targeted proteomics has gained significant popularity in mass spectrometry‐based protein quantification as a method to detect proteins of interest with high sensitivity, quantitative accuracy and reproducibility. However, with the emergence of a wide variety of targeted proteomics methods, some of them with high‐throughput capabilities, it is easy to overlook the essence of each method and to determine what makes each of them a targeted proteomics method. In this viewpoint, we revisit the main targeted proteomics methods and classify them in four categories differentiating those methods that perform targeted data acquisition from targeted data analysis, and those methods that are based on peptide ion data (MS1 targeted methods) from those that rely on the peptide fragments (MS2 targeted methods). 相似文献
5.
The present work firstly described the enantioseparation and determination of flumequine enantiomers in milk, yogurt, chicken, beef, egg, and honey samples by chiral liquid chromatography‐tandem mass spectrometry. The enantioseparation was performed under reversed‐phase conditions on a Chiralpak IC column at 20°C. The effects of chiral stationary phase, mobile phase components, and column temperature on the separation of flumequine enantiomers have been studied in detail. Target compounds were extracted from six different matrices with individual extraction procedure followed by cleanup using Cleanert C18 solid phase extraction cartridge. Good linearity (R2>0.9913) was obtained over the concentration range of 0.125 to 12.5 ng g‐1 for each enantiomer in matrix‐matched standard calibration curves. The limits of detection and limits of quantification of two flumequine enantiomers were 0.015‐0.024 and 0.045‐0.063 ng g‐1, respectively. The average recoveries of the targeted compounds varied from 82.3 to 110.5%, with relative standard deviation less than 11.7%. The method was successfully applied to the determination of flumequine enantiomers in multiple food matrices, providing a reliable method for evaluating the potential risk in animal productions. 相似文献
6.
Introduction – The fruit bodies of Fomes officinalis are used for the treatment of coughs, gastric cancer, rheumatism and hydropsia; however, no method is currently available to assess the quality of this medicinal fungus based on quantitative profile of its main triterpenes. Objective – To develop a simple and accurate HPLC‐UV method for the simultaneous quantification of five lanostane‐type triterpenes in the fruit bodies of F. officinalis. Method – Separations were performed on an Agilent Zorbax Eclipse XDB‐C18 column by gradient elution using acetonitrile : formic acid. Analytes were identified by HPLC coupled with electrospray ionisation mass spectrometry experiments. The quantitative HPLC‐UV method was validated for linearity, precision, accuracy and limits of detection and quantification. Results – Calibration curves presented good linear regression (r > 0.9996) within test ranges. The relative standard deviation of this method was less than 1.7% for intra‐ and inter‐day assays and overall recoveries were 96.4–104.1% for the five compounds analysed. The method was successfully applied to the quantification of five triterpenes in 16 samples of F. officinalis collected from different regions. Conclusion – The developed assay could be considered as a suitable quality control method for F. officinalis. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
7.
Nawal A. Alarfaj Salma A. Al‐tamimi Maha F. El‐Tohamy Maha S. Al‐dughmi 《Luminescence》2019,34(2):222-233
This article suggests a new sequential injection analysis chemiluminescence (SIA‐CL) strategy for monitoring the caffeine (CAF) content in soft and energy drinks using the catalytic activities of different nano‐metal oxides. The present study describes three different SIA‐CL systems (luminol–ferricyanide (III) coupled with Fe2O3 or ZnO nanoparticles (NPs), and luminol–H2O2 coupled with CuONPs. All experimental conditions were optimized and the linear concentration ranges of pure CAF were evaluated using the calibration graphs. The selectivity of the developed SIA‐CL systems was studied under the influence of various interfering species that may be present in soft or energy drinks such as sodium ions, sucrose, glucose, sodium benzoate, sodium citrate, riboflavin, niacin, citric, phosphoric and ascorbic acids. International Council for Harmonization (ICH) guidelines were obeyed for the validation of the suggested CL methods. The developed SIA‐CL systems displayed linear relationships over the concentration ranges 1.0–350, 5.0–400 and 10.0–400 μg ml?1 with Fe2O3 NPs, ZnO NPs and CuO NPs, respectively. The recorded lower limits of detection and quantification were 0.7, 2.7 and 7.8 μg ml?1, and 1.0, 5.0 and 10.0 μg ml?1 for the previously mentioned SIA‐CL systems. The results revealed high selectivity for CAF determination and were in good agreement with those obtained by other reported methods. 相似文献
8.
Deutsch EW Chambers M Neumann S Levander F Binz PA Shofstahl J Campbell DS Mendoza L Ovelleiro D Helsens K Martens L Aebersold R Moritz RL Brusniak MY 《Molecular & cellular proteomics : MCP》2012,11(4):R111.015040
Targeted proteomics via selected reaction monitoring is a powerful mass spectrometric technique affording higher dynamic range, increased specificity and lower limits of detection than other shotgun mass spectrometry methods when applied to proteome analyses. However, it involves selective measurement of predetermined analytes, which requires more preparation in the form of selecting appropriate signatures for the proteins and peptides that are to be targeted. There is a growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptides, but the exchange of this information is hindered by a lack of a standard format. We have developed a new standardized format, called TraML, for encoding transition lists and associated metadata. In addition to introducing the TraML format, we demonstrate several implementations across the community, and provide semantic validators, extensive documentation, and multiple example instances to demonstrate correctly written documents. Widespread use of TraML will facilitate the exchange of transitions, reduce time spent handling incompatible list formats, increase the reusability of previously optimized transitions, and thus accelerate the widespread adoption of targeted proteomics via selected reaction monitoring. 相似文献
9.
Sevgi Tatar Ulu 《Luminescence》2012,27(5):426-430
A sensitive spectrofluorimetric method was developed for the determination of tizanidine in human plasma, urine and pharmaceutical preparations. The method is based on reaction of tizanidine with 1‐dimethylaminonaphthalene‐5‐sulphonyl chloride (dansyl chloride) in an alkaline medium to form a highly fluorescent derivative that was measured at 511 nm after excitation at 383 nm. The different experimental parameters affecting the fluorescence intensity of tizanidine was carefully studied and optimized. The fluorescence–concentration plots were rectilinear over the ranges 50–500 and 20–300 ng/mL for plasma and urine, respectively, detection limits of 1.81 and 0.54 ng/mL and quantification limits of 5.43 and 1.62 ng/mL for plasma and urine, respectively. The method presents good performance in terms of linearity, detection and quantification limits, precision, accuracy and specificity. The proposed method was successfully applied for the determination of tizanidine in pharmaceutical preparations. The results obtained were compared with a reference method, using t‐ and F‐tests. Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
10.
Two simple, selective and accurate methods were developed and validated for the determination of brimonidine tartrate (BT) in pure state and pharmaceutical formulations. Both methods are based on the coupling of the drug with 4‐chloro‐7‐nitro‐2,1,3‐benzoxadiazole in borate buffer (pH 8.5) at 70 °C and measurement of the reaction product spectrophotometrically at 407 nm (method I) or spectrofluorimetrically at 528 nm upon excitation at 460 nm (method II). The calibration graphs were rectilinear over the concentration ranges of 1.0–16.0 and 0.1–4.0 µg/mL with lower detection limits of 0.21 and 0.03, and lower quantification limits of 0.65 and 0.09 µg/mL for methods I and II, respectively. Both methods were successfully applied to the analysis of commercial ophthalmic solution with mean recovery of 99.50 ± 1.00 and 100.13 ± 0.71%, respectively. Statistical analysis of the results obtained by the proposed methods revealed good agreement with those obtained using a comparison method. The proposed spectrofluorimetric method was extended to a stability study of BT under different ICH‐outlined conditions such as alkaline, acidic, oxidative and photolytic degradation. Furthermore, the kinetics of oxidative degradation of the drug was investigated and the apparent first‐order reaction rate constants, half‐life times and Arrhenius equation were estimated. The proposed methods are practical and valuable for routine applications in quality control laboratories for the analysis of BT. Copyright © 2014 John Wiley & Sons, Ltd. 相似文献
11.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,663(2):385-389
A method for the determination of piracetam in human plasma and urine by liquid chromatography with absorbance detection at 206 nm and isocratic elution is proposed. The assay involved a liquid-liquid extraction into hexane-2-propanol at pH 9.2. The calibration graphs were linear in the range 3–40 mg/l in plasma and 100–2000 mg/l in urine. Bias was negligible and coefficients of variation were less than 10% throughout the working range except at 100 mg/l in urine. The limits of quantification were 3 mg/l in plasma and 100 mg/l in urine. The assay was reliably used for pharmacokinetic studies in humans after administration of 800 mg of piracetam per os. 相似文献
12.
So far, mass spectrometry-based targeted proteomics is the most sensitive approach to answer and address specific biological questions in an accurate and quantitative fashion. However, the data analysis design used for such quantification varies in the field leading to discrepancies in the reported values. In this study, different quantification strategies based on calibration curves were evaluated and compared. The best accuracy and coefficient of variation was achieved by ratio to ratio calibration curves. We applied the ratio to ratio quantification approach to analyze very low abundant insulin signaling proteins such as PIK3RA (0.10–0.93 fmol/μg), AKT1 (0.1–0.39 fmol/μg), and the insulin receptor (0.22–2.62 fmol/μg) in a fat cell model and demonstrated the adaptation of this pathway at different states of insulin sensitivity. 相似文献
13.
Systems biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by current proteomics approaches. Selected reaction monitoring (SRM)—also called multiple reaction monitoring—is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions (precursor/fragment ion pairs) in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC‐MS experiment. This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions. Furthermore, normalization and various factors affecting sensitivity and accuracy are discussed. 相似文献
14.
Jeffrey J. Werner A. Celeste Ptak Brian G. Rahm Sheng Zhang Ruth E. Richardson 《Environmental microbiology》2009,11(10):2687-2697
The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole‐sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple‐reaction monitoring (MRM) assays using nano‐liquid chromatography‐tandem mass spectrometry (nLC‐MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R2 > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 ± 1.7 × 103 proteins cell?1 in the KB1TM bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring. 相似文献
15.
Precise protein quantification is essential in comparative proteomics. Currently, quantification bias is inevitable when using proteotypic peptide‐based quantitative proteomics strategy for the differences in peptides measurability. To improve quantification accuracy, we proposed an “empirical rule for linearly correlated peptide selection (ERLPS)” in quantitative proteomics in our previous work. However, a systematic evaluation on general application of ERLPS in quantitative proteomics under diverse experimental conditions needs to be conducted. In this study, the practice workflow of ERLPS was explicitly illustrated; different experimental variables, such as, different MS systems, sample complexities, sample preparations, elution gradients, matrix effects, loading amounts, and other factors were comprehensively investigated to evaluate the applicability, reproducibility, and transferability of ERPLS. The results demonstrated that ERLPS was highly reproducible and transferable within appropriate loading amounts and linearly correlated response peptides should be selected for each specific experiment. ERLPS was used to proteome samples from yeast to mouse and human, and in quantitative methods from label‐free to O18/O16‐labeled and SILAC analysis, and enabled accurate measurements for all proteotypic peptide‐based quantitative proteomics over a large dynamic range. 相似文献
16.
Sergio Pellegrino Franca Sallio Bruno Michele Petrarulo 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1999,729(1-2)
A high-performance liquid chromatographic technique for ethyl alcohol determination in body fluids is proposed. Ethyl alcohol is quantitatively converted into acetaldehyde-phenylhydrazone by oxidation in the presence of alcohol dehydrogenase, nicotinamide–adenine dinucleotide and phenylhydrazine. The derivative is suitable for reversed-phase liquid chromatography and ultraviolet detection at 276 nm. The limits of linearity, detection and quantification as well as accuracy and reproducibility were investigated in water, serum and whole blood. Analytical responses were linear within the 0.008 to 5 g/l range, and the limit of quantification was 0.02 g/l both in aqueous standard and in biological matrix assays. Mean analytical recovery of ethyl alcohol in blood serum averaged 98.2±4.2%, imprecision (CV%) at 0.80 g/l was 2.2%, and the limit of quantification was 0.02 g/l. Serum concentrations of persons that avoided alcoholic beverages for a week were less than the limit of quantification. Ethyl alcohol concentrations in serum and whole blood compared well with those obtained by headspace gas chromatography. This simple and reliable procedure, which was also used for a urine assay, could be suitable for validation of the screening procedures used to monitor ethanol abuse. 相似文献
17.
Advances in the field of targeted proteomics and mass spectrometry have significantly improved assay sensitivity and multiplexing capacity. The high-throughput nature of targeted proteomics experiments has increased the rate of data production, which requires development of novel analytical tools to keep up with data processing demand. Currently, development and validation of targeted mass spectrometry assays require manual inspection of chromatographic peaks from large datasets to ensure quality, a process that is time consuming, prone to inter- and intra-operator variability and limits the efficiency of data interpretation from targeted proteomics analyses. To address this challenge, we have developed TargetedMSQC, an R package that facilitates quality control and verification of chromatographic peaks from targeted proteomics datasets. This tool calculates metrics to quantify several quality aspects of a chromatographic peak, e.g. symmetry, jaggedness and modality, co-elution and shape similarity of monitored transitions in a peak group, as well as the consistency of transitions’ ratios between endogenous analytes and isotopically labeled internal standards and consistency of retention time across multiple runs. The algorithm takes advantage of supervised machine learning to identify peaks with interference or poor chromatography based on a set of peaks that have been annotated by an expert analyst. Using TargetedMSQC to analyze targeted proteomics data reduces the time spent on manual inspection of peaks and improves both speed and accuracy of interference detection. Additionally, by allowing the analysts to customize the tool for application on different datasets, TargetedMSQC gives the users the flexibility to define the acceptable quality for specific datasets. Furthermore, automated and quantitative assessment of peak quality offers a more objective and systematic framework for high throughput analysis of targeted mass spectrometry assay datasets and is a step towards more robust and faster assay implementation. 相似文献
18.
Hannes A Gamper J Peter W Young David L Jones Angela Hodge 《Fungal genetics and biology : FG & B》2008,45(5):581-596
To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan((R)) real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity. 相似文献
19.
Mukesh Kumar Gupta Jin Woo Jung Sang Jun Uhm Hookeun Lee Hoon Taek Lee Kwang Pyo Kim 《Proteomics》2009,9(21):4834-4836
Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay. 相似文献
20.
Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large‐scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide‐binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide‐binding proteins was indeed taking place. Finally, by employing label‐free MS‐based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide‐binding proteome. 相似文献