The development of a powerful new lipidomics tool kit underpinning our objectives

Lead Research Organisation: Babraham Institute
Department Name: UNLISTED

Abstract

Tools for lipidomic analysis of biological extracts have kept pace with their proteomic and DNA-sequencing counterparts. It is now possible to quantify the large majority of lipids and, where relevant, their families of molecular species (ie with different hydrocarbon chains); over 1000 in mammalian cells(126). However, some key problems remain. Most relevant to our ISP has been the difficulty in detecting and quantifying phosphoinositides, particularly the more polar species and their regio-isomers (eg resolving PI(3,4)P2 from PI(4,5)P2 and PI(3,5)P2) and their families of molecular species. This problem is most extreme for PI(3,4,5)P3 and PI(3,4)P2 despite huge interest in their central roles as the output signals from class I PI3Ks. Hence the only approaches that can quantify them cannot resolve molecular species and are low-through-put. This has had 2 major impacts on the field. Firstly, that there is very little data quantifying the primary lipid signals from this pathway PI(3,4)P2 and PIP3, instead many workers use surrogate readouts such as phosphorylation of PKB, and hence there are real problems in trying to understand the dynamics of the pathway as part of the process of developing models to describe its operating principles. Furthermore, it is very difficult to understand the roles and significance of a number of PIP3 and PI(3,4)P2 phosphatases in the pathway because of a lack of these types of measurement. Both of these problems are also hindering pharmaceutical activity in this area. Secondly, it left major conceptual questions about the significance of the distinct molecular species of phosphoinositides. Do they carry any of the PIP3 code? We have shown that chemically synthesized C16:0/C16:0-PIP3 (number of carbon:number of double bonds; dipalmitoyl) was an order of magnitude less potent than C18:0/C20:4-PIP3 (stereoyl/arachidonyl) in activation of PKB in vitro(31). Raising, although not validating, the plausibility of this hypothesis. We have developed a mass-spec-based methodology opening the application of lipidomic strategies to PIP3 and have shown that up to 7 distinct molecular species of PIP3 can be found in cells with a pattern of abundance similar to that of other phosphoinositides, with a predominance of stereoyl/arachidonyl-species(34). Importantly, however, we found that stereoyl/arachidonyl species were further preferentially concentrated in PIP3 compared to the PI(4,5)P2 that acted as the cellular substrate. This must be because the class I PI3Ks or the PIP3-phosphatases preferentially metabolise certain molecular species, either because of differences in the apparent rate constants towards, or access to, the different species. We have hypothesised this is because the molecular species may provide an additional element, “colour”, to the PIP3 code that might be associated with preferential targeting of the lipids into different micro-domains in the plasma membrane known to be enriched in specific proteins. This work is of direct significance to our final high-level objective.

Publications

10 25 50
 
Description Design and establishment of LipidHome: a database for identifying lipid species from mass spectrometry data.
Establishment of nomenclature to define level of accuracy of structural determination from lipid mass spectrometry.
Identification of PI3kinase over activation as a mechanism underpinning overgrowth syndromes.
Demonstration of roles for PI3kinase and phospholipase D in lipid droplet biogenesis and turnover.
Use of isotopomer MS analysis to demonstrate that hypoxia induces a switch from glucose to acetate as a carbon source for lipid biosynthesis through the ACSS2 pathway.
Determination of changes in cellular lipids by infection with HCV and rotaviruses.
Exploitation Route Facilitation of lipid MS through methodological and bioinformatic advances.
Understanding that viral infection impacts upon lipid metabolism.
Sectors Agriculture, Food and Drink,Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Methods we developed have been used by GSK to analyse the results of clinical trials of PI3K inhibitors and by Chiesi in their preclinical evaluation of their PI3K inhibitors in vivo.
First Year Of Impact 2012
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Lipidomic analysis of phosphoinositides 
Description A method to quantify different molecualr species of PIP, PIP2 and PIP3 in small samples of cells or tissues. This has been adopted by groups around the world (eg Japan, France, USA, UK) and by major pharmas R and D centres (eg AZ). 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact The method has been used in analysis of clinical trials and has resulted in a number of excellent publications. The method has been presented at a number of international scientific meetings. 
 
Title Pathway analysis 
Description Development of pathway analysis allowing for identification of enzyme activities modulated by cell stimulation, infection or metabolic change utilising novel bioinformatics methods. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact Identification of potential therapeutic targets to treat infection. 
 
Description AZ/Cosulich 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution We are measuring and modelling agonist and onco-mutant driven changes in PIP3 signalling and then using selective inhibitors and RNAi to understand how the PIP3 signalling network operates
Collaborator Contribution Insights in to commercial directions and interests in this area.
Impact Papers and models (still in development), much work is currently being written-up.
Start Year 2009
 
Description Ben Nichols 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Theoretical contribution upon lipid metabolism and signalling. Lipidomic analysis of cells and tissues.
Collaborator Contribution Generation of flotilla knock outs and experiments upon
Impact paper under review
Start Year 2015
 
Description Cantrell - Measuring PIP3 in lymphocytes 
Organisation University of Dundee
Department Division of Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We used our mass spectrometry methods to measure phosphoinositides in their cells
Collaborator Contribution They provided the cell samples and the intellectual framework for the study
Impact Ross et al (2016) Immunity. 45(3):685-700. doi: 10.1016/j.immuni.2016.07.022 Hukelmann et al (2016) Nat Immunol. 17(1):104-12. doi: 10.1038/ni.3314.
Start Year 2013
 
Description Chiesi 
Organisation Chiesi
Country Italy 
Sector Private 
PI Contribution Measurements of phosphoinositides
Collaborator Contribution Development of PI3K inhibitors towards clinical trials
Impact Multi-disciplinary. We provide lipidomics expertise , Chiesi provide med chem and models
Start Year 2016
 
Description Condliffe - neutrophil biology 
Organisation University of Cambridge
Department School of Clinical Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input, reagents, measurement of phopshoinositides
Collaborator Contribution Intellectual input, personnel, reagents, clinical samples
Impact Angulo et al (2013) Science. 342(6160):866-71. doi: 10.1126/science.1243292 Juss et al (2012) PLoS One. 7(9):e45933. doi: 10.1371/journal.pone.0045933
 
Description Di Christofano_PIP3_mouse_Thyroid 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution Technical expertise: we measured PIP3 by mass spectrometry in their mouse thyroid biopsies
Collaborator Contribution They provided the main intellectual input and the mouse thyroid samples.
Impact Orlacchio A, Ranieri M, Brave M, Arciuch VA, Forde T, De Martino D..... Di Cristofano A, (2017). SGK1 Is a Critical Component of an AKT-Independent Pathway Essential for PI3K-Mediated Tumor Development and Maintenance.. Cancer research, 77 (24), pp. 6914-6926
Start Year 2016
 
Description Divecha 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution Measurement of phosphoinositides. GM mouse projects progressed.
Collaborator Contribution Preparation of cell extracts
Impact Multi-disciplinary, we do lipidomics and mouse work. They do cell line experiments and cancer related experiments
Start Year 2012
 
Description Divecha - phosphoinositides 
Organisation University of Southampton
Department Primary Care and Population Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution expertise, reagents, measurements of phosphoinositides by mass spectrometry
Collaborator Contribution Intellectual framework for study, biological samples
Impact Stijf-Bultsma et al (2015) Mol Cell. 58(3):453-67. doi: 10.1016/j.molcel.2015.03.009. Jones et al (2015) FEBS 281(16):3591-608. doi: 10.1111/febs.12879.
 
Description Doreen Cantrell 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Measurements of phosphoinositides
Collaborator Contribution Samples of lymphocytes
Impact Publications that are fully reported
Start Year 2014
 
Description Floto - Measuring the effect of anti-convulsants on levels of phosphoinositides 
Organisation University of Cambridge
Department Department of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We used our mass spectrometry methods to measure the effect of anti-convulsants on phosphoinositide levels in primary human macrophages
Collaborator Contribution They provided the cell samples
Impact Schiebler et al (2014) EMBO Mol Med. 7(2):127-39. doi: 10.15252/emmm.201404137.
Start Year 2012
 
Description Herve Gillou 
Organisation French National Institute of Agricultural Research
Department INRA laboratory Toxalim
Country France 
Sector Public 
PI Contribution Measurements of Phosphosphoinositides
Collaborator Contribution Preparation of cell samples, dietary manipulation of mice, eg high fat diet
Impact Multi-discipllinary, We perform lipidomics , Toulouse group deal with mouse models and diet manipulation
Start Year 2013
 
Description Ingeborg Hers_ Platelet_PIP3 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution We measured PIP3 in mouse platelet samples and also provided intellectual input into the design of the founding experiments
Collaborator Contribution They were the main intellectual drivers of the project and provided the biological material.
Impact Durrant TN, Hutchinson JL, Heesom KJ, Anderson KE, Stephens LR, Hawkins PT..... Hers I, (2017). In-depth PtdIns(3,4,5)Psignalosome analysis identifies DAPP1 as a negative regulator of GPVI-driven platelet function.. Blood advances, 1 (14), pp. 918-932
Start Year 2014
 
Description Irvine_PI5P4K alpha 
Organisation University of Cambridge
Department Department of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution We measured phosphoinositides by mass spectrometry
Collaborator Contribution They designed the study and provided biological material
Impact Bulley SJ, Droubi A, Clarke JH, Anderson KE, Stephens LR, Hawkins PT, Irvine RF. Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10571-6. doi: 10.1073/pnas.1522478113.
Start Year 2014
 
Description Jane McKeating 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution LPA analysis in cells and tissues , lipidomic analysis of serum and liver tissue
Collaborator Contribution generation of human samples
Impact Publication in J Herpetology
Start Year 2015
 
Description John Walker 
Organisation Medical Research Council (MRC)
Department MRC Mitochondrial Biology Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution lipidomic analysis of protein-lipid complexes
Collaborator Contribution preparation of complexes
Impact joint publication in Open Biology
Start Year 2013
 
Description Kay - PI3K signalling in Dictyostelium 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided intellectual input, reagents and personnel
Collaborator Contribution They provided intellectual input, reagents and personnel
Impact Clark et al (2014) EMBO J. 33(19):2188-200. doi: 10.15252/embj.201488677 Hoeller et al (2013) J Cell Sci. 126(Pt 18):4296-307. doi: 10.1242/jcs.134015.
Start Year 2012
 
Description Parsons - PREX 
Organisation Columbia University Medical Center
Country United States 
Sector Academic/University 
PI Contribution Analysis of phosphoinositides by mass spectrometry
Collaborator Contribution Intellectual frame work for study and biological material
Impact Hodakoski et al (2014) Proc Natl Acad Sci U S A. 111(1):155-60. doi: 10.1073/pnas.1213773111. Epub 2013 Dec 23.
Start Year 2012
 
Description Payastre - Training in the development of mass spectrometry methods for measuring phosphoinositides 
Organisation National Institute of Health and Medical Research (INSERM)
Department Institute of Metabolic and Cardiovascular Disease (I2MC)
Country France 
Sector Public 
PI Contribution We trained a visiting scientist from Toulouse in the use of our mass spectrometry methods for measuring phosphoinositides and helped with the analyses.
Collaborator Contribution They provided cell samples (human platelets) for the analyses
Impact publication: PMID: 29902570
Start Year 2012
 
Description SGK in tumour development 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution We measured phosphoinositide levels by mass spectrometry in mouse tumour samples
Collaborator Contribution They provided the intellectual input and performed most of the experiments
Impact Publications: PMID: 29055016
Start Year 2017
 
Description Sabina Cosulich - Acyl chain composition of phosphoinositides 
Organisation AstraZeneca
Country United Kingdom 
Sector Private 
PI Contribution We have taken the intellectual lead in a collaboration with Dr Sabina Cosulich at AstraZeneca UK to explore the physiological function of the acyl chain compostion of phosphoinositides in mammalian cells.
Collaborator Contribution Sabina Cosulich employed David Barneda on a post-doctoral fellowship at AstraZeneca from 09/16 to 09/19. David split his time working in our lab and AstraZeneca working on this project. We were subsequently awarded a BBSRC grant to employ David Barneda at Babraham from 10/19 - 09/22 to continue this collaboration.
Impact PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K. Malek M, Kielkowska A, Chessa T, Anderson KE, Barneda D, Pir P, Nakanishi H, Eguchi S, Koizumi A, Sasaki J, Juvin V, Kiselev VY, Niewczas I, Gray A, Valayer A, Spensberger D, Imbert M, Felisbino S, Habuchi T, Beinke S, Cosulich S, Le Novère N, Sasaki T, Clark J, Hawkins PT, Stephens LR. Mol Cell. 2017 Nov 2;68(3):566-580.e10. doi: 10.1016/j.molcel.2017.09.024. Epub 2017 Oct 19. How is the acyl chain composition of phosphoinositides created and does it matter? Barneda D, Cosulich S, Stephens L, Hawkins P. Biochem Soc Trans. 2019 Oct 31;47(5):1291-1305. doi: 10.1042/BST20190205. Follow on funding: BBSRC BB/T002530/1 Multidisciplinary: mass spectrometry, organic chemistry, cell biology
Start Year 2016
 
Description Sasaki_ mass spec and prostate 
Organisation Akita University
Country Japan 
Sector Academic/University 
PI Contribution Intellectual input, reagents, mass spectrometric methods, personnel
Collaborator Contribution Intellectual input, reagents, mass spectrometric methods, personnel
Impact Norton et al (2016) Adv Biol Regul. 60:36-45. doi: 10.1016/j.jbior.2015.10.005 Ferguson et al (2007) Nat Cell Biol. 9(1):86-91. Malek M, Kielkowska A, Chessa T, Anderson KE, Barneda D, Pir P..... Stephens LR, (2017). PTEN Regulates PI(3,4)PSignaling Downstream of Class I PI3K.. Molecular cell, 68 (3), pp. 566-580.e10
 
Description Van haesebroeck - PI3K 
Organisation University College London
Department UCL Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input, reagents and mass spectrometric analysis of phosphoinositides
Collaborator Contribution Intellectual input, biological samples. Wide ranging collaboration covering several projects.
Impact Alliouachene et al (2015) Cell Rep. 13(9):1881-94. doi: 10.1016/j.celrep.2015.10.052 Gyori et al (2014) Arthritis Rheumatol. 66(8):2210-21. doi: 10.1002/art.38660. Kulkarni et al (2011) Sci Signal. 4(168):ra23. doi: 10.1126/scisignal.2001617.
Start Year 2009
 
Description Volker Haucke 
Organisation Leibniz Association
Department Leibniz-Institute for Molecular Pharmacology
Country Germany 
Sector Academic/University 
PI Contribution Measurements of phopshoinositides and advice about assays for inostiol phosphates
Collaborator Contribution They have established assays of intracellualr trafficking using high content imaging technology and to measure mTOR1 distribution and roles.
Impact Multi-disciplinary, high content imaging, mass spectrometry, biochemisty
Start Year 2015
 
Description Williams - PI3K 
Organisation Medical Research Council (MRC)
Department MRC Laboratory of Molecular Biology (LMB)
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input, reagents, personnel
Collaborator Contribution Intellectual input, reagents, personnel
Impact Angulo et al (2013) Science 342(6160):866-71. doi: 10.1126/science.1243292 Zhang et al (2011) Mol Cell. 41(5):567-78. doi: 10.1016/j.molcel.2011.01.026 Houslay et al (2016) Sci Signal. 9(441):ra82. doi: 10.1126/scisignal.aae0453 Krugmann et al (2004) Curr Biol.14(15):1380-4
 
Description Williams_Impact of deleting inositol 3-phosphatase in Dictyostelium 
Organisation Royal Holloway, University of London
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution We measured phosphoinositides by mass spectrometry
Collaborator Contribution They designed the project and provided biological material
Impact Frej et al (2016) Mol Cell Biol. 36(10):1464-79. doi: 10.1128/MCB.00039-16. Print 2016 May 15.
Start Year 2013
 
Description Ageing research working group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact working group aiming to define the physiological society's policy on research into ageing and the societal consequences. This was used to inform the Department of Health and the public.
Year(s) Of Engagement Activity 2018,2019
 
Description Invited lecturer to international meetings (average 2-3 per year) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Generated discussion, exchange of ideas and collaborations
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Invited lecturer to international meetings (average 2-3 per year) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact promoted discussions, collaborations

scientific collaborations, joint grants and publications
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Lipidomics workshops and lipid databasing, standards and nomenclature 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Establishing internationally agreed format for reporting lipid mass spectrometry data, agreeing structures for archiving of lipidomics data, international links, transfer of LIPID MAPS website and databasing capability from USA to Babraham and Cardiff
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019
 
Description Public feedback exercise 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact The Signalling Department, in parallel with other BI departments, presented our research to a professionally selected sample of the UK public with the interntion of gaining public feedback on our institute and research.
Year(s) Of Engagement Activity 2015
 
Description Schools day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 125 pupils attended schools day, talks given to all and lab practicals ran for two small groups.
Year(s) Of Engagement Activity 2018,2019
 
Description Training other researchers in use of lipidomics techniques. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Researchers visit BI for 1-5days typically to observe, discuss or trouble shoot lipidomics techniques. PhD students, PIs, technicians and commercial staff have been involved. Researchers from Japan, France and UK.
Year(s) Of Engagement Activity 2012,2013,2014,2015