1,721,180 research outputs found
Combining amine metabolomics and quantitative proteomics of cancer cells using derivatization with isobaric tags
No full textQuantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons
Time-resolved proximity labeling of protein networks associated with ligand-activated EGFR
No full textLigand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases
Comprehensive temporal protein dynamics during the diauxic shift in saccharomyces cerevisiae
No full textYeast (Saccharomyces cerevisiae) has served as a key model system in biology and as a benchmark for “omics” technology. Although near-complete proteomes of log phase yeast have been measured, protein abundance in yeast is dynamic, particularly during the transition from log to stationary phase. Defining the dynamics of proteomic changes during this transition, termed the diauxic shift, is important to understand the basic biology of proliferative versus quiescent cells. Here, we perform temporal quantitative proteomics to fully capture protein induction and repression during the diauxic shift. Accurate and sensitive quantitation at a high temporal resolution and depth of proteome coverage was achieved using TMT10 reagents and LC-MS3 analysis on an Orbitrap Fusion tribrid mass spectrometer deploying synchronous precursor selection. Triplicate experiments were analyzed using the time-course R package and a simple template matching strategy was used to reveal groups of proteins with similar temporal patterns of protein induction and repression. Within these groups are functionally distinct types of proteins such as those of glyoxylate metabolism and many proteins of unknown function not previously associated with the diauxic shift (e.g. YNR034W-A and FMP16). We also perform a dual time-course experiment to determine Hap2-dependent proteins during the diauxic shift. These data serve as an important basic model for fermentative versus respiratory growth of yeast and other eukaryotes and are a benchmark for temporal quantitative proteomics.
The yeast proteome serves as a valuable model in systems biology (1) and as such has been used to gauge technological milestones in proteomics. In recent years, the depth of protein identification in logarithmically-growing yeast has expanded to near-comprehensiveness (>4000 identified proteins) (2–3). However, the yeast proteome is dynamic, and understanding regulatory networks requires a comprehensive grasp of the timing of induction or repression of specific sets of proteins. Proteome dynamics in yeast depend largely on substrate availability, the major of which is glucose. The diauxic shift, the transition from log phase growth on glucose to stationary phase upon glucose exhaustion, involves temporal coordination of protein regulation that is still not completely understood. Because log phase yeast resemble fermentative cancer cells, the diauxic shift is also considered important basic model for understanding the requirements of proliferative cell growth. As such, a landmark gene expression study of the diauxic shift at the transcript level (4) has served as an important resource in biology (5). However, despite recent progress, the dynamic nature of the diauxic shift at the proteome level has not been adequately explored. Doing so requires the ability to perform comprehensive quantitative proteomic analysis with a sufficient number of time-points to resolve the timing of protein induction or repression. Although temporal proteomic data changes have recently been reported within both log and stationary phase (6) using six-plex quantitative proteomics with TMT, and during the transition between log and stationary phase using a four-plex Neucode strategy, highly resolved proteomic data able to fully capture protein dynamics between the two states are lacking.
Several recent advances in mass spectrometry and multiplexed quantitative proteomics potentiate accurate measurements of highly resolved time courses at a comprehensive depth of proteome coverage. First, tandem mass tag technology (TMT)1 has been expanded to compare up to 10 samples simultaneously (TMT10) using isotopologue-containing reporter ions distinguishable by high-resolution mass spectrometry (7). TMT reagents work on the principle of isobaric tagging whereby their addition to a peptide maintains a nominal mass, but cleavable reporter ions are used to compare peptide abundance between samples (8). Second, although isobaric reporter ion tagging strategies have been plagued by interference from co-isolated peptides, using MS3 scans for reporter ion quantitation has been shown to ensure quantitative accuracy (9). Third, the MS3 approach has recently been enhanced by techniques to isolate multiple MS2 precursors (SPS) for greater MS3 scan sensitivity (10). These advancements, deployed on the Orbitrap Fusion tribrid mass spectrometer allow high temporal resolution (10 time points), proteomic depth (>4500 proteins), and quantitative accuracy (11). We here aimed to fully capture the intricate temporal proteome dynamics over the full diauxic shift at a comprehensive level using 10-plex TMT and LC-MS3.
The data we present here extend current understanding of protein dynamics during diauxic shift in yeast by virtue of the high temporal resolution and comprehensive depth achieved. Data were collected in triplicate allowing us to assess temporal significance, following which we applied a simple template-profile matching strategy to map the timing of induction or repression of proteins of specific functions in relation to glucose exhaustion. The data set also resolves the timing of the induction of many proteins of unknown function (e.g. FMP16, YNR034W-A, and TMA17) as well those not previously associated with the transition to stationary phase (e.g. YKL065W-A and PAI3). In an additional, dual time-course experiment, we show both high reproducibility between analyses of different strains and provide a putative list of proteins dependent on the respiratory growth-related transcription factor, Hap2. These include many known Hap2-dependent proteins such as those of glyoxylate metabolism and oxidative phosphorylation as well as several novel Hap2-dependent proteins supporting carnitine metabolism (e.g. AGP2). These data serve as a benchmark in temporal quantitative proteomics and are a valuable resource for the biological community in understanding fermentative versus respiratory metabolism in eukaryotic cells
Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase
Full text linkThe hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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