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Lazar C.,University Grenoble Alpes | Lazar C.,CEA Grenoble | Lazar C.,French Institute of Health and Medical Research | Gatto L.,Computational Proteomics Unit | And 11 more authors.
Journal of Proteome Research | Year: 2016

Missing values are a genuine issue in label-free quantitative proteomics. Recent works have surveyed the different statistical methods to conduct imputation and have compared them on real or simulated data sets and recommended a list of missing value imputation methods for proteomics application. Although insightful, these comparisons do not account for two important facts: (i) depending on the proteomics data set, the missingness mechanism may be of different natures and (ii) each imputation method is devoted to a specific type of missingness mechanism. As a result, we believe that the question at stake is not to find the most accurate imputation method in general but instead the most appropriate one. We describe a series of comparisons that support our views: For instance, we show that a supposedly "under-performing" method (i.e., giving baseline average results), if applied at the "appropriate" time in the data-processing pipeline (before or after peptide aggregation) on a data set with the "appropriate" nature of missing values, can outperform a blindly applied, supposedly "better-performing" method (i.e., the reference method from the state-of-the-art). This leads us to formulate few practical guidelines regarding the choice and the application of an imputation method in a proteomics context. © 2016 American Chemical Society.


Lim K.,Harvard University | Groen A.,Cambridge Center for Proteomics | Molostvov G.,University of Warwick | Lu T.,Harvard University | And 10 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2015

Context: α-Klotho has emerged as a powerful regulator of the aging process. To date, the expression profile of α-Klotho in human tissues is unknown, and its existence in some human tissue types is subject to much controversy. Objective: This is the first study to characterize systemwide tissue expression of transmembrane α-Klotho in humans. We have employed next-generation targeted proteomic analysis using parallel reaction monitoring in parallel with conventional antibody-based methods to determine the expression and spatial distribution of human α-Klotho expression in health. Results: The distribution of α-Klotho in human tissues from various organ systems, including arterial, epithelial, endocrine, reproductive, and neuronal tissues, was first identified by immunohistochemistry. Kidney tissues showed strong α-Klotho expression, whereas liver did not reveal a detectable signal. These results were next confirmed by Western blotting of both whole tissues and primary cells. To validate our antibody-based results, α-Klotho-expressing tissues were subjected to parallel reaction monitoring mass spectrometry (data deposited at ProteomeXchange, PXD002775) identifying peptides specific for the full-length, transmembrane α-Klotho isoform. Conclusions: The data presented confirmα-Klotho expression in the kidney tubule and in the artery and provide evidence of α-Klotho expression across organ systems and cell types that has not previously been described in humans. Copyright © 2015 by the Endocrine Society.


Anjum R.S.,University of Cambridge | Bray S.M.,University of Cambridge | Blackwood J.K.,University of Cambridge | Kilkenny M.L.,University of Cambridge | And 11 more authors.
Nature Communications | Year: 2015

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved. © 2015 Macmillan Publishers Limited. All rights reserved.


Wang M.,Tennis Court Road | Bond N.J.,Cambridge Center for Proteomics | Letcher A.J.,Tennis Court Road | Richardson J.P.,Tennis Court Road | And 4 more authors.
Biochemical Journal | Year: 2010

PtdIns5P 4-kinases IIα and IIβ are cytosolic and nuclear respectively when transfected into cells, including DT40 cells [Richardson, Wang, Clarke, Patel and Irvine (2007) Cell. Signalling 19, 1309-1314]. In the present study we have genomically tagged both type II PtdIns5P 4-kinase isoforms in DT40 cells. Immunoprecipitation of either isoform from tagged cells, followed by MS, revealed that they are associated directly with each other, probably by heterodimerization. We quantified the cellular levels of the type II PtdIns5P 4-kinase mRNAs by real-time quantitative PCR and the absolute amount of each isoform in immunoprecipitates by MS using selective reaction monitoring with 14N,13C-labelled internal standard peptides. The results suggest that the dimerization is complete and random, governed solely by the relative concentrations of the two isoforms. Whereas PtdIns5P 4-kinase IIβ is >95% nuclear, as expected, the distribution of PtdIns4P 4-kinase IIα is 60% cytoplasmic (all bound to membranes) and 40% nuclear. In vitro, PtdIns5P 4-kinase IIα was 2000-fold more active as a PtdIns5P 4-kinase than the IIβ isoform. Overall the results suggest a function of PtdIns5P 4-kinase IIβ may be to target the more active IIα isoform into the nucleus. © The Authors.


Nikolovski N.,University of Cambridge | Nikolovski N.,Cambridge Center for Proteomics | Shliaha P.V.,University of Cambridge | Shliaha P.V.,Cambridge Center for Proteomics | And 6 more authors.
Plant Physiology | Year: 2014

The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (dataindependent acquisition method using ion mobility separation known as LC-IMS-MSE [or HDMSE]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle. © 2014 American Society of Plant Biologists. All rights reserved.


Batalha I.L.,New University of Lisbon | Zhou H.,Cambridge Center for Proteomics | Lilley K.,Cambridge Center for Proteomics | Lowe C.R.,University of Cambridge | Roque A.C.A.,New University of Lisbon
Journal of Chromatography A | Year: 2016

Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins (α- and β-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides. © 2016 Elsevier B.V.


PubMed | Cambridge Center for Proteomics, University of Cambridge and New University of Lisbon
Type: | Journal: Journal of chromatography. A | Year: 2016

Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins (- and -caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides.


Swatton J.E.,Tennis Court Road | Swatton J.E.,Cancer Research UK Research Institute | Davenport P.W.,Tennis Court Road | Maunders E.A.,Tennis Court Road | And 4 more authors.
PLoS ONE | Year: 2016

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and1H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12-13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system. © Copyright 2016 Swatton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


PubMed | Cambridge Center for Proteomics, Max Planck Institute for Terrestrial Microbiology and University of Cambridge
Type: | Journal: Nature communications | Year: 2015

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.


PubMed | Tennis Court Road and Cambridge Center for Proteomics
Type: Journal Article | Journal: PloS one | Year: 2016

The macrolide antibiotic, azithromycin (AZM), has been reported to improve the clinical outcome of cystic fibrosis patients, many of whom are chronically-infected with Pseudomonas aeruginosa. However, the highest clinically-achievable concentrations of this drug are well-below the minimum inhibitory concentration for P. aeruginosa, raising the question of why AZM exhibits therapeutic activity. One possibility that has been raised by earlier studies is that AZM inhibits quorum sensing (QS) by P. aeruginosa. To explicitly test this hypothesis the changes brought about by AZM treatment need to be compared with those associated with specific QS mutants grown alongside in the same growth medium, but this has not been done. In this work, we used quantitative 2D-difference gel electrophoresis and 1H-NMR spectroscopy footprint analysis to examine whether a range of clinically-relevant AZM concentrations elicited proteomic and metabolomic changes in wild-type cultures that were similar to those seen in cultures of defined QS mutants. Consistent with earlier reports, over half of the AZM-induced spot changes on the 2D gels were found to affect QS-regulated proteins. However, AZM modulated very few protein spots overall (compared with QS) and collectively, these modulated proteins comprised only a small fraction (12-13%) of the global QS regulon. We conclude that AZM perturbs a sub-regulon of the QS system but does not block QS per se. Reinforcing this notion, we further show that AZM is capable of attenuating virulence factor production in another Gram-negative species that secretes copious quantities of exoenzymes (Serratia marcescens), even in the absence of a functional QS system.

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