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Albert J. R. Heck - One of the best experts on this subject based on the ideXlab platform.
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Mass spectrometry-based Quantitative Proteomics
Expert Review of Proteomics, 2020Co-Authors: Albert J. R. Heck, Jeroen KrijgsveldAbstract:A major aim of present-day Proteomics is to study changes in protein expression levels at a global level, ideally monitoring all proteins present in cells or tissue. Mass spectrometry is a well-respected technology in Proteomics that is widely used for the identification of proteins. More recently, methodologies have been introduced showing that mass spectrometry can also be used for protein quantification. This article reviews various mass spectrometry-based technologies in Quantitative Proteomics, highlighting several interesting applications in areas ranging from cell biology to clinical applications.
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benchmarking stable isotope labeling based Quantitative Proteomics
Journal of Proteomics, 2013Co-Authors: A. F.maarten Altelaar, Christian K Frese, Marco L Hennrich, A W Schram, H T M Timmers, Christian Preisinger, Albert J. R. Heck, Shabaz MohammedAbstract:Abstract Several Quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or chemical isotope labeling with MS based quantification or chemical labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale Quantitative Proteomics experiments, comparing SILAC, dimethyl and TMT labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and chemical labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
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applications of stable isotope dimethyl labeling in Quantitative Proteomics
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Duangnapa Kovanich, Shabaz Mohammed, Salvatore Cappadona, Reinout Raijmakers, Arjen Scholten, Albert J. R. HeckAbstract:Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in Quantitative Proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions.
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multiplex peptide stable isotope dimethyl labeling for Quantitative Proteomics
Nature Protocols, 2009Co-Authors: Paul J Boersema, Reinout Raijmakers, Shabaz Mohammed, Simone Lemeer, Albert J. R. HeckAbstract:Accurate quantification of protein expression in biological systems is an increasingly important part of Proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput Proteomics experiments. Although alternative multiplex Quantitative Proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60–90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5–3 days to complete.
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An experimental correction for arginine-to-proline conversion artifacts in SILAC-based Quantitative Proteomics
Nature Methods, 2007Co-Authors: D. Van Hoof, Albert J. R. Heck, Martijn W. H. Pinkse, D. Ward-van Oostwaard, Christine L. Mummery, Jeroen KrijgsveldAbstract:An experimental correction for arginine-to-proline conversion artifacts in SILAC-based Quantitative Proteomics
Ruedi Aebersold - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Proteomics: challenges and opportunities in basic and applied research
Nature Protocols, 2017Co-Authors: Olga T Schubert, Hannes L Röst, Ben C Collins, George Rosenberger, Ruedi AebersoldAbstract:In this Perspective, we discuss developments in mass-spectrometry-based proteomic technology over the past decade from the viewpoint of our laboratory. We also reflect on existing challenges and limitations, and explore the current and future roles of Quantitative Proteomics in molecular systems biology, clinical research and personalized medicine. In their Perspective, Schubert et al . discuss developments and challenges in mass-spectrometry-based Proteomics technology in the past decade and explore its role in molecular systems biology, clinical research and personalized medicine.
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Selected reaction monitoring for Quantitative Proteomics: A tutorial
Molecular Systems Biology, 2008Co-Authors: Vinzenz Lange, Bruno Domon, Paola Picotti, Ruedi AebersoldAbstract: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.
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Quantitative Proteomics by Stable Isotope Labeling and Mass Spectrometry
Methods of Molecular Biology, 2007Co-Authors: Ruedi AebersoldAbstract:The goal of Quantitative Proteomics is to systematically study static state or perturbation-induced changes in protein profile. Most of the recently developed mass spectrometry (MS)-based Quantitative proteomic methods employ stable isotope labeling to introduce signature mass tags to peptides/proteins that can be used by a mass spectrometer to quantify each analyte and to determine the sample from which it originates. In this chapter, we discuss several methods for the introduction of mass tags to proteins and peptides for MS-based Quantitative proteomic analysis, including isotope-coded affinity tags, stable isotope labeling by amino acids in cell culture, global internal standard technology, and mass-coded abundance tagging.
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eLS - Quantitative Proteomics (ICAT
Encyclopedia of Life Sciences, 2006Co-Authors: Timothy J. Griffin, Jamie Sherman, Ruedi AebersoldAbstract:An essential aspect of proteomic analysis is the identification and quantification of each protein present in two or more complex mixtures. The use of isotope-coded affinity tag (ICAT™) reagents and mass spectrometry forms part of Quantitative proteomic analysis technology. Keywords: Quantitative Proteomics; mass spectrometry; liquid chromatography; two-dimensional gel electrophoresis
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Quantitative Proteomics of cerebrospinal fluid from patients with alzheimer disease
Journal of Alzheimer's Disease, 2005Co-Authors: Jing Zhang, Ruedi Aebersold, Dave R Goodlett, Joseph F Quinn, Elaine R Peskind, Jeffrey Kaye, Yong Zhou, Eugene C Yi, Qin Wang, Thomas J MontineAbstract:Biomarkers to assist in the diagnosis and medical management of Alzheimer disease (AD) are a pressing need. We have employed a proteomic approach, microcapillary liquid chromatography mass spectrometry of proteins labeled with isotope-coded affinity tags (ICAT), to quantify relative changes in the proteome of human cerebrospinal fluid (CSF) obtained from the lumbar cistern. Using CSF from well-characterized AD patients and age-matched controls at 2 different institutions, we quantified protein concentration ratios of 42% of the 390 CSF proteins that we have identified and found differences > or = 20% in over half of them. We confirmed our findings by western blot and validated this approach by quantifying relative levels of amyloid precursor protein and cathepsin B in 17 AD patients and 16 control individuals. Quantitative Proteomics of CSF from AD patients compared to age-matched controls, as well as from other neurodegenerative diseases, will allow us to generate a roster of proteins that may serve as specific biomarker panels for AD and other geriatric dementias.
Alec C Kimmelman - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Proteomics identifies ncoa4 as the cargo receptor mediating ferritinophagy
Nature, 2014Co-Authors: Joseph D Mancias, Xiaoxu Wang, Steven P Gygi, Wade J Harper, Alec C KimmelmanAbstract:Through a Quantitative Proteomics analysis, a cohort of proteins is identified that associate with autophagosomes, among them a new cargo receptor called NCOA4 that, in response to iron deprivation, targets ferritin to autophagosomes and thereby releases iron.
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Quantitative Proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy
Nature, 2014Co-Authors: Joseph D Mancias, J. Wade Harper, Xiaoxu Wang, Stephen P. Gygi, Alec C KimmelmanAbstract:Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo, including organelles, proteins or intracellular pathogens, are targeted for selective autophagy is limited. Here we use Quantitative Proteomics to identify a cohort of novel and known autophagosome-enriched proteins in human cells, including cargo receptors. Like known cargo receptors, nuclear receptor coactivator 4 (NCOA4) was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species but is degraded via autophagy to release iron through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin led to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy), which is critical for iron homeostasis, and provides a resource for further dissection of autophagosomal cargo-receptor connectivity.
Matthias Mann - One of the best experts on this subject based on the ideXlab platform.
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Quantitative Proteomics for Epigenetics
ChemBioChem, 2011Co-Authors: H. Christian Eberl, Matthias Mann, Michiel VermeulenAbstract:Mass spectrometry has made many contributions to the chromatin field through the mapping of histone modifications and the identification of protein complexes involved in gene regulation. MS-based Proteomics has now evolved from the identification of single protein spots in gels to the identification and quantification of thousands of proteins in complex mixtures. Quantitative approaches also allow comparative and time-resolved analysis of post-translational modifications. An important emerging field is the unbiased interaction analysis of proteins with other proteins, defined protein modifications, specific DNA and RNA sequences, and small molecules. Quantitative Proteomics can also accurately monitor whole proteome changes in response to perturbation of the gene expression machinery. We provide an up-to-date review of modern Quantitative proteomic technology and its applications in the field of epigenetics.
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use of stable isotope labeling by amino acids in cell culture as a spike in standard in Quantitative Proteomics
Nature Protocols, 2011Co-Authors: Tamar Geiger, Jacek R Wisniewski, Sara Zanivan, Yasushi Ishihama, Marcus Kruger, Matthias MannAbstract:Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in Quantitative Proteomics
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stable isotope labeling by amino acids in cell culture silac applied to Quantitative Proteomics of bacillus subtilis
Journal of Proteome Research, 2010Co-Authors: Boumediene Soufi, Chanchal Kumar, Florian Gnad, Ivan Mijakovic, Matthias Mann, Boris MacekAbstract:We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale Quantitative Proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic for lysine and high accuracy mass spectrometry for downstream analysis, we identified and quantified changes in the levels of more than 1500 proteins in each of the tested conditions with high biological and technical reproducibility. With a total of 1928 identified proteins, this study presents one of the most comprehensive Quantitative Proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under phosphate starvation, demonstrating the full compatibility of the method with site-specific detection and quantitation of...
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functional and Quantitative Proteomics using silac
Nature Reviews Molecular Cell Biology, 2006Co-Authors: Matthias MannAbstract:Stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful format for Quantitative Proteomics. What are the current applications for SILAC? And, how will this technology be used in the future?
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mass spectrometric based approaches in Quantitative Proteomics
Methods, 2003Co-Authors: Leonard J Foster, Matthias MannAbstract:Classically, experiments aimed at studying changes in protein expression have always followed a small set of proteins. This focused approach was necessary since tools to efficiently analyze large numbers of proteins were simply not available. Large-scale Quantitative Proteomics promises to produce reams of data that previously would have taken decades to measure with classical methods. Mass spectrometry is already a well-established protein identification tool and recent methodological developments indicate that it can also be successfully applied to extract Quantitative data of protein abundance. From the first reports 4 years ago, numerous schemes to take advantage of stable isotope nuclei incorporation in proteins and peptides have been developed. Here we review the benefits and pitfalls of some of the most commonly used protocols, focusing on a procedure now being used extensively in our laboratory, stable isotope labeling with amino acids in cell culture (SILAC). The basic theory, application, and data analysis of a SILAC experiment are discussed. The emerging nature of these techniques and the rapid pace of technological development make forecasting the directions of the field difficult but we speculate that SILAC will soon be a key tool of Quantitative Proteomics.
Shabaz Mohammed - One of the best experts on this subject based on the ideXlab platform.
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benchmarking stable isotope labeling based Quantitative Proteomics
Journal of Proteomics, 2013Co-Authors: A. F.maarten Altelaar, Christian K Frese, Marco L Hennrich, A W Schram, H T M Timmers, Christian Preisinger, Albert J. R. Heck, Shabaz MohammedAbstract:Abstract Several Quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or chemical isotope labeling with MS based quantification or chemical labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale Quantitative Proteomics experiments, comparing SILAC, dimethyl and TMT labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and chemical labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
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applications of stable isotope dimethyl labeling in Quantitative Proteomics
Analytical and Bioanalytical Chemistry, 2012Co-Authors: Duangnapa Kovanich, Shabaz Mohammed, Salvatore Cappadona, Reinout Raijmakers, Arjen Scholten, Albert J. R. HeckAbstract:Mass spectrometry has proven to be an indispensable tool for protein identification, characterization, and quantification. Among the possible methods in Quantitative Proteomics, stable isotope labeling by using reductive dimethylation has emerged as a cost-effective, simple, but powerful method able to compete at any level with the present alternatives. In this review, we briefly introduce experimental and software methods for proteome analysis using dimethyl labeling and provide a comprehensive overview of reported applications in the analysis of (1) differential protein expression, (2) posttranslational modifications, and (3) protein interactions.
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multiplex peptide stable isotope dimethyl labeling for Quantitative Proteomics
Nature Protocols, 2009Co-Authors: Paul J Boersema, Reinout Raijmakers, Shabaz Mohammed, Simone Lemeer, Albert J. R. HeckAbstract:Accurate quantification of protein expression in biological systems is an increasingly important part of Proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput Proteomics experiments. Although alternative multiplex Quantitative Proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60–90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5–3 days to complete.
