Accurate quantitation of standard peptides used for quantitative proteomics.
Ontology highlight
ABSTRACT:
SUBMITTER: Bordeerat NK
PROVIDER: S-EPMC2766299 | biostudies-literature | 2009 Aug
REPOSITORIES: biostudies-literature
Publications
Accurate quantitation of standard peptides used for quantitative proteomics.
Bordeerat Narisa K NK Georgieva Nadia I NI Klapper David G DG Collins Leonard B LB Cross Tyra J TJ Borchers Christoph H CH Swenberg James A JA Boysen Gunnar G
Proteomics 20090801 15
MS-based proteomics has become an indispensable tool in system biology generating a need for accurate and precise quantitation of peptide standards. The presented method utilizes ultra performance LC-MS/MS (UPLC-MS/MS) to accurately quantify peptide standards at concentrations of 0.1-10 microM. The ability for accurate quantitation of micro-molar concentrations has the advantages that quantitation can be performed routinely with high precision and the high sensitivity of the method minimizes the ...[more]
Similar Datasets
Project description:PurposeTargeted proteomics using MRM with stable-isotope-labeled internal-standard (SIS) peptides is the current method of choice for protein quantitation in complex biological matrices. Better quantitation can be achieved with the internal standard-addition method, where successive increments of synthesized natural form (NAT) of the endogenous analyte are added to each sample, a response curve is generated, and the endogenous concentration is determined at the x-intercept. Internal NAT-addition, however, requires multiple analyses of each sample, resulting in increased sample consumption and analysis time.Experimental designTo compare the following three methods, an MRM assay for 34 high-to-moderate abundance human plasma proteins is used: classical internal SIS-addition, internal NAT-addition, and external NAT-addition-generated in buffer using NAT and SIS peptides. Using endogenous-free chicken plasma, the accuracy is also evaluated.ResultsThe internal NAT-addition outperforms the other two in precision and accuracy. However, the curves derived by internal vs. external NAT-addition differ by only ?3.8% in slope, providing comparable accuracies and precision with good CV values.Conclusions and clinical relevanceWhile the internal NAT-addition method may be "ideal", this new external NAT-addition can be used to determine the concentration of high-to-moderate abundance endogenous plasma proteins, providing a robust and cost-effective alternative for clinical analyses or other high-throughput applications.
| S-EPMC6084352 | biostudies-literature
Project description:A method to achieve accurate measurement of unmetabolized volatile organic compounds (VOCs) in urine was developed and characterized. The method incorporates a novel preanalytical approach of adding isotopically labeled internal standard (ISTD) analogues directly to the collection container at the point of collection to compensate for analyte loss to the headspace and the collection container surfaces. Using this approach, 45 toxic VOCs ranging in water solubility and boiling point were evaluated and analyzed by headspace solid-phase microextraction/gas chromatography-mass spectrometry. Results show that urine VOCs could be equally lost to the container headspace as to the container surface suggesting similarity of these two regions as partition phases. Surface adsorption loss was found to trend with compound water solubility. In particular, with no headspace, more nonpolar VOCs experienced substantial losses (e.g., 48% for hexane) in a standard 120 mL urine cup at concentrations in the low- and sub-ppb range. The most polar VOCs evaluated (e.g., tetrahydrofuran) showed no significant loss. Other commonly practiced methods for urine sample collection and analysis such as aliquoting, specimen freezing, and use of surrogate ISTD were found to significantly bias results. With this method, we achieved errors ranging from -8.0 to 4.8% of spiked urine specimens. Paired urine and blood specimens from cigarette smokers were compared to assess this method.
| S-EPMC8154218 | biostudies-literature
Project description:Proenkephalin (PE) represents the precursor protein of the active peptide neurotransmitter enkephalin. Quantitative analysis of peptides and proteins is an objective of mass spectrometry-based studies of biological systems and will be important for studying the proteolytic conversion of proproteins to active enkephalin and neuropeptides. The goal of this study was to define and optimize quantitation of different amounts of tryptic peptides derived from PE using light (H4, 4 hydrogens) and heavy (D4, 4 deuteriums) succinic anhydride for isotopic labeling of peptides analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparisons were made between PE-derived peptides with and without internal standards. Importantly, incorporation of internal standards of known amounts of heavy isotope-labeled tryptic peptides of PE provided linear calibration plots with accurate quantitation. In contrast, comparison of light and heavy isotope-labeled peptides without internal standards produced variable and inaccurate nonlinear isotopic ratio comparisons of PE-derived peptides. These results demonstrate that use of internal standards composed of a defined amount(s) of standard peptides (PE-derived tryptic peptides) is necessary for high-quality linear quantitation of peptides by isotopic labeling and MS/MS.
| S-EPMC2731681 | biostudies-other
Project description:Genome-wide localization of chromatin and transcription regulators can be detected by a variety of techniques. Here, we describe a novel method 'greenCUT&RUN' for genome-wide profiling of transcription regulators, which has a very high sensitivity, resolution, accuracy and reproducibility, whilst assuring specificity. Our strategy begins with tagging of the protein of interest with GFP and utilizes a GFP-specific nanobody fused to MNase to profile genome-wide binding events. By using a GFP-nanobody the greenCUT&RUN approach eliminates antibody dependency and variability. Robust genomic profiles were obtained with greenCUT&RUN, which are accurate and unbiased towards open chromatin. By integrating greenCUT&RUN with nanobody-based affinity purification mass spectrometry, 'piggy-back' DNA binding events can be identified on a genomic scale. The unique design of greenCUT&RUN grants target protein flexibility and yields high resolution footprints. In addition, greenCUT&RUN allows rapid profiling of mutants of chromatin and transcription proteins. In conclusion, greenCUT&RUN is a widely applicable and versatile genome-mapping technique.
| S-EPMC8136828 | biostudies-literature
Project description:A major scientific challenge at the present time for cancer research is the determination of the underlying biological basis for cancer development. It is further complicated by the heterogeneity of cancer's origin. Understanding the molecular basis of cancer requires studying the dynamic and spatial interactions among proteins in cells, signaling events among cancer cells, and interactions between the cancer cells and the tumor microenvironment. Recently, it has been proposed that large-scale protein expression analysis of cancer cell proteomes promises to be valuable for investigating mechanisms of cancer transformation. Advances in mass spectrometry technologies and bioinformatics tools provide a tremendous opportunity to qualitatively and quantitatively interrogate dynamic protein-protein interactions and differential regulation of cellular signaling pathways associated with tumor development. In this review, progress in shotgun proteomics technologies for examining the molecular basis of cancer development will be presented and discussed.
| S-EPMC2352161 | biostudies-other
Project description:Droplet digital PCR provides superior accuracy in nucleic acid quantitation. The requirement of microfluidics to generate and analyze the emulsions, however, is a barrier to its adoption, particularly in low resource or clinical settings. Here, we report a novel method to prepare ddPCR droplets by vortexing and readout the results by bulk analysis of recovered amplicons. We demonstrate the approach by accurately quantitating SARS-CoV-2 sequences using entirely bulk processing and no microfluidics. Our approach for quantitating reactions should extend to all digital assays that generate amplicons, including digital PCR and LAMP conducted in droplets, microchambers, or nanoliter wells. More broadly, our approach combines important attributes of ddPCR, including enhanced accuracy and robustness to inhibition, with the high-volume sample processing ability of quantitative PCR.
| S-EPMC7814815 | biostudies-literature
Project description:Changes in plasmalogen glycerophosphoethanolamine (PE-P) composition (structure and abundance) are a key indicator of altered lipid metabolism. Differential changes in the levels of PE-P have been reported in different disease states, including neurodegenerative diseases. Of particular interest, traumatic brain injury (TBI) has resulted in altered expression of glycerophospholipid profiles, including PE-P. To date, most analytical assays assessing PE-P have focused on general lipidomic workflows to evaluate the relative, semi-quantitative abundance of PE-P during disease progression. This approach provides a broad evaluation of PE-P, yet often lacks specificity and sensitivity for individual PE-P structures which is a necessity for robust quantitative data. The present study highlights the development of a targeted, quantitative method using a HILIC separation and selective reaction monitoring mass spectrometry for the confident identification and accurate quantitation of PE-P. Our innovative method incorporates both the sn-1 alkyl vinyl ether and sn-2 acyl chain as product ion transitions, for specific and sensitive quantitation of 100 PE-P structures. Our method also uniquely allowed for the unambiguous assignment and quantitation of di-unsaturated sn-1 PE-P structures, which to date have not been conclusively quantified. Application of this assay to a TBI mouse model resulted in distinct temporal profiles for plasma PE-P up to 28 days post injury. Plasma PE-P were significantly increased 24 h after induced TBI, followed by a gradual reduction to sham concentrations by day 28. Overall, we established a structure-specific, quantitative assay for identification and quantitation of a comprehensive set of PE-P structures with demonstrated relevance to brain injury.
| S-EPMC8581249 | biostudies-literature
Project description:Multiplexed quantitative analyses of complex proteomes enable deep biological insight. While a multitude of workflows have been developed for multiplexed analyses, the most quantitatively accurate method (SPS-MS3) suffers from long acquisition duty cycles. We built a new, real-time database search (RTS) platform, Orbiter, to combat the SPS-MS3 method's longer duty cycles. RTS with Orbiter eliminates SPS-MS3 scans if no peptide matches to a given spectrum. With Orbiter's online proteomic analytical pipeline, which includes RTS and false discovery rate analysis, it was possible to process a single spectrum database search in less than 10 ms. The result is a fast, functional means to identify peptide spectral matches using Comet, filter these matches, and more efficiently quantify proteins of interest. Importantly, the use of Comet for peptide spectral matching allowed for a fully featured search, including analysis of post-translational modifications, with well-known and extensively validated scoring. These data could then be used to trigger subsequent scans in an adaptive and flexible manner. In this work we tested the utility of this adaptive data acquisition platform to improve the efficiency and accuracy of multiplexed quantitative experiments. We found that RTS enabled a 2-fold increase in mass spectrometric data acquisition efficiency. Orbiter's RTS quantified more than 8000 proteins across 10 proteomes in half the time of an SPS-MS3 analysis (18 h for RTS, 36 h for SPS-MS3).
| S-EPMC7295121 | biostudies-literature
Project description:The mzQuantML standard has been developed by the Proteomics Standards Initiative for capturing, archiving and exchanging quantitative proteomic data, derived from mass spectrometry. It is a rich XML-based format, capable of representing data about two-dimensional features from LC-MS data, and peptides, proteins or groups of proteins that have been quantified from multiple samples. In this article we report the development of an open source Java-based library of routines for mzQuantML, called the mzqLibrary, and associated software for visualising data called the mzqViewer. The mzqLibrary contains routines for mapping (peptide) identifications on quantified features, inference of protein (group)-level quantification values from peptide-level values, normalisation and basic statistics for differential expression. These routines can be accessed via the command line, via a Java programming interface access or a basic graphical user interface. The mzqLibrary also contains several file format converters, including import converters (to mzQuantML) from OpenMS, Progenesis LC-MS and MaxQuant, and exporters (from mzQuantML) to other standards or useful formats (mzTab, HTML, csv). The mzqViewer contains in-built routines for viewing the tables of data (about features, peptides or proteins), and connects to the R statistical library for more advanced plotting options. The mzqLibrary and mzqViewer packages are available from https://code.google.com/p/mzq-lib/.
| S-EPMC4973685 | biostudies-literature
Project description:Structure-specific characterization and quantitation is often required for effective functional studies of oligosaccharides. Inside the gut, HMOs are preferentially bound and catabolized by the beneficial bacteria. HMO utility by these bacteria employs structure-specific catabolism based on a number of glycosidases. Determining the activity of these enzymes requires accurate quantitation of a large number of structures. In this study, we describe a method for the quantitation of human milk oligosaccharide (HMO) structures employing LC/MS and isotopically labeled internal standards. Data analysis was accomplished with a newly developed software tool, LC/MS Searcher, that employs a reference structure library to process LC/MS data yielding structural identification with accurate quantitation. The method was used to obtain a meta-enzyme analysis of bacteria, the simultaneous characterization of all glycosidases employed by bacteria for the catabolism of milk oligosaccharides. Analysis of consumed HMO structures confirmed the utility of a ?-1,3-galactosidase in Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis). In comparison, Bifidobacterium breve ATCC 15700 showed significantly less HMO catabolic activity compared to B. infantis.
| S-EPMC3457638 | biostudies-literature