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Ruedi Aebersold - One of the best experts on this subject based on the ideXlab platform.
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chapter 4 getting absolute determining absolute protein quantities via Selected Reaction Monitoring mass spectrometry
2014Co-Authors: Christina Ludwig, Ruedi AebersoldAbstract:Accurate quantification of proteins is important for a wide range of questions in molecular and cell biology, systems biology, or clinical research. Depending on the specific question asked, either relative quantitative changes across multiple samples (relative quantification) or absolute protein concentrations of proteins in a particular sample (absolute quantification) are required. Absolute quantification is beneficial, for example, in studies on protein complex stoichiometries, mathematical modeling of biological processes, clinical biomarker development, or for comprehensive inter-experimental, inter-laboratory and inter-organism comparisons. In recent years, targeted mass spectrometry via Selected Reaction Monitoring (SRM) has proven suitable for reproducible, precise and sensitive absolute quantification of predetermined sets of proteins. In this chapter we provide a general overview of the most commonly applied absolute quantification strategies with SRM. These include the use of stable-isotope-labeled peptide and protein standards, as well as label-free strategies. Advantages and limitations of each workflow are presented and compared. Finally, important challenges and pitfalls specific for absolute protein quantification are highlighted and future perspectives for the field of targeted proteomics are discussed.
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Automated Selected Reaction Monitoring data analysis workflow for large-scale targeted proteomic studies
Nature Protocols, 2013Co-Authors: Silvia Surinova, Lucia Espona, Ruth Hüttenhain, Olga Vitek, Ching-yun Chang, Ruedi AebersoldAbstract:Targeted proteomics based on Selected Reaction Monitoring (SRM) mass spectrometry is commonly used for accurate and reproducible quantification of protein analytes in complex biological mixtures. Strictly hypothesis-driven, SRM assays quantify each targeted protein by collecting measurements on its peptide fragment ions, called transitions. To achieve sensitive and accurate quantitative results, experimental design and data analysis must consistently account for the variability of the quantified transitions. This consistency is especially important in large experiments, which increasingly require profiling up to hundreds of proteins over hundreds of samples. Here we describe a robust and automated workflow for the analysis of large quantitative SRM data sets that integrates data processing, statistical protein identification and quantification, and dissemination of the results. The integrated workflow combines three software tools: mProphet for peptide identification via probabilistic scoring; SRMstats for protein significance analysis with linear mixed-effect models; and PASSEL, a public repository for storage, retrieval and query of SRM data. The input requirements for the protocol are files with SRM traces in mzXML format, and a file with a list of transitions in a text tab-separated format. The protocol is especially suited for data with heavy isotope-labeled peptide internal standards. We demonstrate the protocol on a clinical data set in which the abundances of 35 biomarker candidates were profiled in 83 blood plasma samples of subjects with ovarian cancer or benign ovarian tumors. The time frame to realize the protocol is 1-2 weeks, depending on the number of replicates used in the experiment.
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proteome wide Selected Reaction Monitoring assays for the human pathogen streptococcus pyogenes
Nature Communications, 2012Co-Authors: Christofer Karlsson, Ruedi Aebersold, Lars Malmstrom, Johan MalmströmAbstract:Selected Reaction Monitoring mass spectrometry (SRM-MS) is a targeted proteomics technology used to identify and quantify proteins with high sensitivity, specificity and high reproducibility. Execution of SRM-MS relies on protein-specific SRM assays, a set of experimental parameters that requires considerable effort to develop. Here we present a proteome-wide SRM assay repository for the gram-positive human pathogen group A Streptococcus. Using a multi-layered approach we generated SRM assays for 10,412 distinct group A Streptococcus peptides followed by extensive testing of the Selected Reaction Monitoring assays in >200 different group A Streptococcus protein pools. Based on the number of SRM assay observations we created a rule-based Selected Reaction Monitoring assay-scoring model to select the most suitable assays per protein for a given cellular compartment and bacterial state. The resource described here represents an important tool for deciphering the group A Streptococcus proteome using Selected Reaction Monitoring and we anticipate that concepts described here can be extended to other pathogens.
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Selected Reaction Monitoring based proteomics workflows potential pitfalls and future directions
Nature Methods, 2012Co-Authors: Paola Picotti, Ruedi AebersoldAbstract:In this Review, the authors provide a guide through to the different steps involved in Selected Reaction Monitoring as well as discuss its applications.
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Selected Reaction Monitoring based proteomics workflows potential pitfalls and future directions
Nature Methods, 2012Co-Authors: Paola Picotti, Ruedi AebersoldAbstract:Selected Reaction Monitoring (SRM) is a targeted mass spectrometry technique that is emerging in the field of proteomics as a complement to untargeted shotgun methods. SRM is particularly useful when predetermined sets of proteins, such as those constituting cellular networks or sets of candidate biomarkers, need to be measured across multiple samples in a consistent, reproducible and quantitatively precise manner. Here we describe how SRM is applied in proteomics, review recent advances, present Selected applications and provide a perspective on the future of this powerful technology.
Pierre Yves Communal - One of the best experts on this subject based on the ideXlab platform.
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multiresidue analysis of multiclass pesticides in lavandin essential oil by lc ms ms using the scheduled Selected Reaction Monitoring mode
Analytical Chemistry, 2011Co-Authors: Yoann Fillâtre, David Rondeau, Alain Jadashecart, Brice Bonnet, Antoine Daguin, Pierre Yves CommunalAbstract:In this paper we describe the development of the first multiclass pesticide residue method applied to essential oils. A total of 70 pesticides covering a wide range of polarity and currently used on essential oil crops have been included in the method. The procedure consists of a 10-fold dilution of lavandin essential oil followed by a direct injection analysis by liquid chromatography/tandem mass spectrometry. The system used is an API 4000 QTrap equipped with an electrospray ionization interface and operating in scheduled Selected Reaction Monitoring acquisition mode. Matrix effects were evaluated by comparing the slopes of matrix-matched and solvent-based calibration curves. Weak signal suppression or enhancement (<20%) was observed for most of the compounds. Method sensitivity was determined statistically by the injection of five matrix-matched calibration curves with the distribution’s normality and the variance’s homogeneity checked before establishment of a suitable regression model. Limits of dete...
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advantages of the scheduled Selected Reaction Monitoring algorithm in liquid chromatography electrospray ionization tandem mass spectrometry multi residue analysis of 242 pesticides a comparative approach with classical Selected Reaction Monitoring m
Rapid Communications in Mass Spectrometry, 2010Co-Authors: Yoann Fillâtre, David Rondeau, Pierre Yves Communal, Alain JadashecartAbstract:This paper illustrates the advantages of using the scheduled Selected Reaction Monitoring (sSRM) algorithm available in Analyst® Software 1.5 to build SRM acquisition methods in the application field of pesticide multi-residue analysis. The principle is to monitor the SRM transitions only when necessary. Based on the analytes' retention times, the scheduled SRM algorithm decreases the number of concurrent SRM transitions monitored at any point in time, allowing both cycle time and dwell time to remain optimal at higher levels of SRM multiplexing. To compare the scheduled SRM and the classical SRM modes, a mixture containing 242 multi-class pesticides has been analyzed ten times by three acquisition methods, using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with an API 4000 QTrap™ mass spectrometer. The scheduled SRM mode demonstrates better results in all fields: more data points per peak, better reproducibility (coefficients of variation (CVs) <5%) and higher signal-to-noise ratio (S/N), even when the number of SRM transitions is doubled. The use of scheduled SRM mode instead of the classical one gives an enhancement of the limits of quantification by a factor two or even higher (up to six), depending on the analyte transitions. Copyright © 2010 John Wiley & Sons, Ltd.
Eleftherios P. Diamandis - One of the best experts on this subject based on the ideXlab platform.
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Targeted Selected Reaction Monitoring mass spectrometric immunoassay for insulin-like growth factor 1.
PloS one, 2013Co-Authors: Amol Prakash, Scott Peterman, Bryan Krastins, Vathany Kulasingam, Eric E. Niederkofler, David A. Phillips, Urban A. Kiernan, Kemmons A. Tubbs, Eleftherios P. Diamandis, Mary F. LopezAbstract:Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a Selected Reaction Monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.
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quantitative analysis of energy metabolic pathways in mcf 7 breast cancer cells by Selected Reaction Monitoring assay
Molecular & Cellular Proteomics, 2012Co-Authors: Andrei P Drabovich, Maria P Pavlou, Apostolos Dimitromanolakis, Eleftherios P. DiamandisAbstract:To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were Selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex Selected Reaction Monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.
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Development of a multiplex Selected Reaction Monitoring assay for quantification of biochemical markers of down syndrome in amniotic fluid samples.
Journal of proteome research, 2012Co-Authors: Eduardo Martínez-morillo, Andrei P Drabovich, Chan-kyung J. Cho, Julie L.v. Shaw, Antoninus Soosaipillai, Eleftherios P. DiamandisAbstract:Down syndrome (DS) is one of the most common chromosomal abnormalities affecting about 1 of every 700 fetuses. Current screening strategies have detection rates of 90−95% at a 5% false positive rate. The aim of this study was to discover new biomarkers of DS in amniotic fluid by using a multiplex Selected Reaction Monitoring assay. Nine proteins were analyzed: CEL, CPA1, MUC13, CLCA1, MUC5AC, PLUNC, and HAPLN1, and CGB as positive control and serotransferrin as negative control. One proteotypic peptide for each protein was Selected, and internal heavy isotope-labeled peptide standards were spiked into the samples. Fifty-four samples from pregnant women carrying normal (n = 37) or DS-affected (n = 17) fetuses were analyzed. The median protein concentrations for DS and normal samples, respectively, were as follows: 20 and 49 ng/mL (p 0.05) for PLUNC; 144 and 86 ng/mL (p 0.05) for serotransferrin. Statistically significant differences were found in six out of the seven candidate proteins analyzed, reflecting a different regulation in DS.
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verification of male infertility biomarkers in seminal plasma by multiplex Selected Reaction Monitoring assay
Molecular & Cellular Proteomics, 2011Co-Authors: Andrei P Drabovich, Keith Jarvi, Eleftherios P. DiamandisAbstract:Seminal plasma is a promising biological fluid to use for noninvasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex Selected Reaction Monitoring assay and measured the relative abundance of 31 proteins in 30 seminal plasma samples from normal, nonobstructive azoospermia and post-vasectomy individuals. Median levels of some proteins were decreased by more than 100-fold in nonobstructive azoospermia or post-vasectomy samples, in comparison with normal samples. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to reanalyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1–1000 μg/ml but were significantly decreased in nonobstructive azoospermia and post-vasectomy. These data allowed us to select, for the first time, biomarkers to discriminate between normal, nonobstructive azoospermia, and post-vasectomy (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, and SPAG11B) performed with absolute or nearly absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in nonobstructive azoospermia seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay that can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.
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verification of a biomarker discovery approach for detection of down syndrome in amniotic fluid via multiplex Selected Reaction Monitoring srm assay
Journal of Proteomics, 2011Co-Authors: Andrei P Drabovich, Eleftherios P. Diamandis, Ihor BatruchAbstract:Prenatal screening test for Down syndrome (DS) can be improved by discovery of novel biomarkers. A multiplex Selected Reaction Monitoring (SRM) assay was developed to test previously identified thirteen candidate proteins in amniotic fluid (AF). One unique peptide was Selected for each protein based on discovery data, while three MS/MS transitions were Selected based on intelligent SRM results. For one of the candidates, matrix metalloproteinase-2 (MMP2), ELISA was also performed to validate SRM results in AF and to test serum samples. Comparison of AF samples from DS versus controls via SRM assay revealed five proteins that were differentially expressed. Bile salt-activated lipase, mucin-13, carboxypeptidase A1, and dipeptidyl peptidase 4 showed a decrease in DS-affected AF, and MMP2 showed an increase, in comparison to controls (P<0.05). Discovery-based spectral counting ratios and SRM ratios showed a strong correlation, and MMP2 ELISA further confirmed the validity of the SRM data. Potential implications of differentially expressed proteins during fetal development are proposed. Our data also shows that SRM can provide a high-throughput and accurate platform for biomarker verification.
Yoann Fillâtre - One of the best experts on this subject based on the ideXlab platform.
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multiresidue analysis of multiclass pesticides in lavandin essential oil by lc ms ms using the scheduled Selected Reaction Monitoring mode
Analytical Chemistry, 2011Co-Authors: Yoann Fillâtre, David Rondeau, Alain Jadashecart, Brice Bonnet, Antoine Daguin, Pierre Yves CommunalAbstract:In this paper we describe the development of the first multiclass pesticide residue method applied to essential oils. A total of 70 pesticides covering a wide range of polarity and currently used on essential oil crops have been included in the method. The procedure consists of a 10-fold dilution of lavandin essential oil followed by a direct injection analysis by liquid chromatography/tandem mass spectrometry. The system used is an API 4000 QTrap equipped with an electrospray ionization interface and operating in scheduled Selected Reaction Monitoring acquisition mode. Matrix effects were evaluated by comparing the slopes of matrix-matched and solvent-based calibration curves. Weak signal suppression or enhancement (<20%) was observed for most of the compounds. Method sensitivity was determined statistically by the injection of five matrix-matched calibration curves with the distribution’s normality and the variance’s homogeneity checked before establishment of a suitable regression model. Limits of dete...
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advantages of the scheduled Selected Reaction Monitoring algorithm in liquid chromatography electrospray ionization tandem mass spectrometry multi residue analysis of 242 pesticides a comparative approach with classical Selected Reaction Monitoring m
Rapid Communications in Mass Spectrometry, 2010Co-Authors: Yoann Fillâtre, David Rondeau, Pierre Yves Communal, Alain JadashecartAbstract:This paper illustrates the advantages of using the scheduled Selected Reaction Monitoring (sSRM) algorithm available in Analyst® Software 1.5 to build SRM acquisition methods in the application field of pesticide multi-residue analysis. The principle is to monitor the SRM transitions only when necessary. Based on the analytes' retention times, the scheduled SRM algorithm decreases the number of concurrent SRM transitions monitored at any point in time, allowing both cycle time and dwell time to remain optimal at higher levels of SRM multiplexing. To compare the scheduled SRM and the classical SRM modes, a mixture containing 242 multi-class pesticides has been analyzed ten times by three acquisition methods, using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with an API 4000 QTrap™ mass spectrometer. The scheduled SRM mode demonstrates better results in all fields: more data points per peak, better reproducibility (coefficients of variation (CVs) <5%) and higher signal-to-noise ratio (S/N), even when the number of SRM transitions is doubled. The use of scheduled SRM mode instead of the classical one gives an enhancement of the limits of quantification by a factor two or even higher (up to six), depending on the analyte transitions. Copyright © 2010 John Wiley & Sons, Ltd.
Maria Teresa Galceran - One of the best experts on this subject based on the ideXlab platform.
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fast liquid chromatography tandem mass spectrometry highly selective Selected Reaction Monitoring for the determination of toltrazuril and its metabolites in food
Analytical and Bioanalytical Chemistry, 2010Co-Authors: Anna Martinezvillalba, Claudia P.b. Martins, Encarnación Moyano, Maria Teresa GalceranAbstract:In this work a fast liquid chromatography (LC)–tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF3 (toltrazuril and toltrazuril sulfone) and CHF3• (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C18 Fused-Core™ column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective Selected Reaction Monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a cleanup with a C18 cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 µg kg-1).
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liquid chromatography tandem mass spectrometry highly selective Selected Reaction Monitoring for the analysis of isopropylthioxanthone in packaged food
Journal of Chromatography A, 2008Co-Authors: Hector Gallartayala, Encarnación Moyano, Maria Teresa GalceranAbstract:Isopropylthioxanthone (ITX), usually applied as a mixture of 2- and 4-isomers, is a common photo-initiator in UV inks used in paper- or plastic-based packaging materials. In this work a pentafluorophenylpropyl column (HS F5) has been used to achieve the chromatographic separation of the two isomers. A gradient elution with acetonitrile and a 25mM formic acid-ammonium formate at pH 3.75 are required to provide an Rs of 1.3 between the two compounds. The fragmentation pattern of ITX was studied using two mass analyzers, an ion trap (IT) (multi-stage fragmentation) and a triple quadrupole mass analyzer of hyperbolic rods (accurate mass (AM) measurement). The protonated molecule [M+H](+) observed in the mass spectrometry (MS) spectrum lost an isopropyl group, [M+H-C(3)H(6)](+). Later, this ion fragmented, yielding the radical ion [M+H-C(3)H(6)-CHO](+). The elemental composition of these product ions was confirmed by AM measurement. Electrospray ionization (ESI) was used as an ionization source to couple liquid chromatography (LC) to MS. Instrumental quality parameters of three acquisition modes provided by the triple quadrupole mass analyzer were studied and good run-to-run precision (relative standard deviation, RSD, lower than 10%) and limits of detection (LODs) down to 0.8pg injected in the LC-MS/MS system were obtained. Finally the LC-MS/MS method using H-SRM Q1 acquisition mode was used to analyze 2- and 4-ITX in a range of food samples. The use of highly selective Selected Reaction Monitoring (H-SRM on Q1) resulted in improved selectivity without sensitivity loss.
