Refined Preparation and Use of Anti-diglycine Remnant (K-ε-GG) Antibody Enables Routine Quantification of 10,000s of Ubiquitination Sites in Single Proteomics Experiments
01 Mar 2013-Molecular & Cellular Proteomics (American Society for Biochemistry and Molecular Biology)-Vol. 12, Iss: 3, pp 825-831
TL;DR: A number of improvements to the K-ε-GG enrichment workflow are described, including optimized antibody and peptide input requirements, antibody cross-linking, and improved off-line fractionation prior to enrichment.
About: This article is published in Molecular & Cellular Proteomics.The article was published on 2013-03-01 and is currently open access. It has received 289 citations till now.
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TL;DR: Using quantitative proteomics, it is found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKzF3, by the CRBN-CRL4 ubiquitin ligase, which are essential transcription factors in multiple myeloma.
Abstract: Lenalidomide is a drug with clinical efficacy in multiple myeloma and other B cell neoplasms, but its mechanism of action is unknown. Using quantitative proteomics, we found that lenalidomide causes selective ubiquitination and degradation of two lymphoid transcription factors, IKZF1 and IKZF3, by the CRBN-CRL4 ubiquitin ligase. IKZF1 and IKZF3 are essential transcription factors in multiple myeloma. A single amino acid substitution of IKZF3 conferred resistance to lenalidomide-induced degradation and rescued lenalidomide-induced inhibition of cell growth. Similarly, we found that lenalidomide-induced interleukin-2 production in T cells is due to depletion of IKZF1 and IKZF3. These findings reveal a previously unknown mechanism of action for a therapeutic agent: alteration of the activity of an E3 ubiquitin ligase, leading to selective degradation of specific targets.
1,254 citations
TL;DR: This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.
Abstract: We report a mass spectrometry-based method for the integrated analysis of protein expression, phosphorylation, ubiquitination and acetylation by serial enrichments of different post-translational modifications (SEPTM) from the same biological sample. This technology enabled quantitative analysis of nearly 8,000 proteins and more than 20,000 phosphorylation, 15,000 ubiquitination and 3,000 acetylation sites per experiment, generating a holistic view of cellular signal transduction pathways as exemplified by analysis of bortezomib-treated human leukemia cells.
538 citations
TL;DR: In this overview for the special PTM issue of Molecular and Cellular Proteomics, it is taken stock of where MS-based proteomics stands in the large-scale analysis of protein modifications.
510 citations
Cites background from "Refined Preparation and Use of Anti..."
...sites can be specifically enriched and analyzed in a generic workflow (19, 20)....
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TL;DR: A scalable mono‐allelic strategy for profiling the HLA peptidome is implemented and a strategy for systematically learning the rules of endogenous antigen presentation is demonstrated, providing an updated portrait of antigen processing rules.
477 citations
Cites result from "Refined Preparation and Use of Anti..."
...The count of ubiquitination sites (previously observed in KG-1, Jurkat, or MM1S cells (Krönke et al., 2015; Krönke et al., 2014; Udeshi et al., 2012, 2013), was positively associated with HLA-peptide presentation, consistent with the known role for ubiquitin in delivering proteins to the…...
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TL;DR: This work states that with the ability to quantitatively measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - the authors are now entering a new era in metabolism research, with mass spectrometry (MS)-based proteomics at the helm.
Abstract: Metabolism research is undergoing a renaissance because many diseases are increasingly recognized as being characterized by perturbations in intracellular metabolic regulation. Metabolic changes can be conferred through changes to the expression of metabolic enzymes, the concentrations of substrates or products that govern reaction kinetics, or post-translational modification (PTM) of the proteins that facilitate these reactions. On the 60th anniversary since its discovery, reversible protein phosphorylation is widely appreciated as an essential PTM regulating metabolism. With the ability to quantitatively measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - we are now entering a new era in metabolism research, with mass spectrometry (MS)-based proteomics at the helm.
383 citations
References
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TL;DR: By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
Abstract: DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
31,015 citations
TL;DR: MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data, detects peaks, isotope clusters and stable amino acid isotope–labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space and achieves mass accuracy in the p.p.b. range.
Abstract: Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
12,340 citations
"Refined Preparation and Use of Anti..." refers methods in this paper
...K- -GG site identification and quantification information were obtained from the MaxQuant Gly-Gly sites and evidence tables....
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...MS Data Analysis—MS data were analyzed using the MaxQuant (11, 12) software version 1.3.0.5 and searched against the human Uniprot database containing 81,470 entries....
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...For the SILAC-based benchmarking experiments, site level information was obtained directly from the MaxQuant Gly-Gly sites table....
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...A list of 248 common laboratory contaminants provided by MaxQuant was also added to the database....
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...MS Data Analysis—MS data were analyzed using the MaxQuant (11, 12) software version 1....
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TL;DR: The hierarchical model of Lonnstedt and Speed (2002) is developed into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples and the moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom.
Abstract: The problem of identifying differentially expressed genes in designed microarray experiments is considered. Lonnstedt and Speed (2002) derived an expression for the posterior odds of differential expression in a replicated two-color experiment using a simple hierarchical parametric model. The purpose of this paper is to develop the hierarchical model of Lonnstedt and Speed (2002) into a practical approach for general microarray experiments with arbitrary numbers of treatments and RNA samples. The model is reset in the context of general linear models with arbitrary coefficients and contrasts of interest. The approach applies equally well to both single channel and two color microarray experiments. Consistent, closed form estimators are derived for the hyperparameters in the model. The estimators proposed have robust behavior even for small numbers of arrays and allow for incomplete data arising from spot filtering or spot quality weights. The posterior odds statistic is reformulated in terms of a moderated t-statistic in which posterior residual standard deviations are used in place of ordinary standard deviations. The empirical Bayes approach is equivalent to shrinkage of the estimated sample variances towards a pooled estimate, resulting in far more stable inference when the number of arrays is small. The use of moderated t-statistics has the advantage over the posterior odds that the number of hyperparameters which need to estimated is reduced; in particular, knowledge of the non-null prior for the fold changes are not required. The moderated t-statistic is shown to follow a t-distribution with augmented degrees of freedom. The moderated t inferential approach extends to accommodate tests of composite null hypotheses through the use of moderated F-statistics. The performance of the methods is demonstrated in a simulation study. Results are presented for two publicly available data sets.
11,864 citations
"Refined Preparation and Use of Anti..." refers methods in this paper
...T-statistic calculated from the limma package in the R environment as described previously (3, 13)....
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TL;DR: SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system and is applied to the relative quantitation of changes in protein expression during the process of muscle cell differentiation.
5,653 citations
TL;DR: A novel peptide search engine using a probabilistic scoring model that can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, and accommodates extremely large databases.
Abstract: A key step in mass spectrometry (MS)-based proteomics is the identification of peptides in sequence databases by their fragmentation spectra. Here we describe Andromeda, a novel peptide search engine using a probabilistic scoring model. On proteome data, Andromeda performs as well as Mascot, a widely used commercial search engine, as judged by sensitivity and specificity analysis based on target decoy searches. Furthermore, it can handle data with arbitrarily high fragment mass accuracy, is able to assign and score complex patterns of post-translational modifications, such as highly phosphorylated peptides, and accommodates extremely large databases. The algorithms of Andromeda are provided. Andromeda can function independently or as an integrated search engine of the widely used MaxQuant computational proteomics platform and both are freely available at www.maxquant.org. The combination enables analysis of large data sets in a simple analysis workflow on a desktop computer. For searching individual spect...
4,689 citations
"Refined Preparation and Use of Anti..." refers methods in this paper
...K- -GG site identification and quantification information were obtained from the MaxQuant Gly-Gly sites and evidence tables....
[...]
...MS Data Analysis—MS data were analyzed using the MaxQuant (11, 12) software version 1.3.0.5 and searched against the human Uniprot database containing 81,470 entries....
[...]
...For the SILAC-based benchmarking experiments, site level information was obtained directly from the MaxQuant Gly-Gly sites table....
[...]
...A list of 248 common laboratory contaminants provided by MaxQuant was also added to the database....
[...]
...MS Data Analysis—MS data were analyzed using the MaxQuant (11, 12) software version 1....
[...]