Mark Bennett is the lab manager of the MS facilities for quantitative analysis within the Department of Life Sciences.

Our analytical equipment includes both LC-MS/MS and GC-MS platforms.

The facility handles a very wide range of projects, covering both proteins and small molecules. Recently we have been focussing on methods for targeted quantitative measurements of tryptic peptides (“Mass Westerns”) as an alternative to using antibodies for quantifying proteins. Further examples of the types of analysis available are listed below.

  • Quantitative proteomics including targeted analysis and quantitative studies of post translational modifications. 
  • Analysis of plant hormones from Arabidopsis and other species.
  • Sugar analysis from plants and aphids.
  • Analysis of glucosinolates and their degradation products.
  • Fatty acid profiling of microbes and plants.
  • DNA/RNA oxidation in microbes.
  • Analysis of cyclic dinucleotides in bacteria.
  • Proteomic analysis of Arabidopsis and other plants.

details

LCMS

The core LC-MS systems comprises of two highly flexible ABSCIEX QTrap LCMS instruments. The QTrap MS is a hybrid Triple Quadrupole/Ion Trap mass spectrometer that can be used in several modes depending on the application.

Our most sensitive instrument, the latest 6500 model, was recently purchased with college SIF funding; the MS is coupled to an Eksigent microflow LC system to provide a very sensitive but robust platform for high throughput analysis of peptides and small molecules.

An older 2000QTrap is available for the analysis of molecules where the greatest sensitivity is not required, this can coupled to Agilent binary or capillary LC systems.

Several other standalone LC systems are available these are equipped with a range of detectors (UV, RI,DAD).

How it works

The MS has three quadrupoles: Q1 and Q3 are used as mass filters; Q2 is a collision cell where intact molecules and fragments from Q1 are broken into fragments of smaller masses.

Quadropoles

Triple Quad modes

MRM (multiple reaction monitoring) - Q1 and Q3 fixed: Q3 set to detect only one particular mass that is a known fragment (daughter) from the intact molecule (parent) of known mass filtered through Q1.

This is the standard quantitative mode used for targeted metabolite and protein analysis.

Advantages: high sensitivity, high reproducibility, low noise, simultaneous measurement of up to (1000) transitions.

Disadvantages: Only works for known molecules, requires standard compound for full optimisation

Precursor ion scanning - Q3 fixed Q1 scans
Looks for all possible molecules containing a common sub-structure of known mass, an example would be for looking for phosphopeptides in a tryptic digest of a phosphorulated protein by looking for ions showing loss of phosphate.

Neutral loss - Q1 and Q3 scan with a fixed mass difference
Looks for loss of a common fragment occurring in a range of parent compounds.

 

Ion Trap modes

Enhanced MS (EMS) - Q1 is set to “open” so no mass filtering. Full scan spectra are generated from scans out of Q3 .This is usually used to give an overview of  all of the ions in sample.

Enhanced production (EPI) - Q1 is set to a fixed mass (m/z value), usually based on a known molecule. As with EMS, spectra are generated from scans out of Q3. Advantage is to generate full scan spectra from a single parent ion e.g. for peptide sequencing. Sensitivity can approach that achieved via MRM, but EPI provides much more spectral information.

Enhanced resolution (ER) - More accurate m/z determination (but still not as accurate as some other LCMS systems such as QTOF).

Mixed modes (quadrupole/ion trap) are possible. This can be data dependent, for example, a signal from an MRM scan could trigger an EPI scan to confirm a compound identity. Another example would be the selection of only doubly charged molecules to trigger an EPI, as in the analysis of tryptic peptide fragments.

 

For further information or to discuss access, please contact mhbennett@imperial.ac.uk

GCMS

For GC-MS we operate two Agilent 6890/5973 systems.

The two 6890/5973 Agilent GC/MS systems are single quadrupole instruments. Both systems are set up for split/splitless injection with capillary GC, and operate in electron impact (EI) mode. One of the GCs is additionally equipped with a FID detector, the other has an OPTIC-2 thermal desorption(TD) inlet. TD is largely used for analysis of highly volatile compounds, collected as “headspace” samples adsorbed onto a chemical trap. Biological volatiles include attractants, deterrents and signalling compounds

For further information or to discuss access, please contact mhbennett@imperial.ac.uk

Publications

2014

 

  • Schumacher J, Waite CJ, Bennett MH, Perez MF, Shethi K, Buck M.Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics. Front Plant Sci 5:242 2014 Author URL
  • Lougheed KE, Bennett MH, Williams HDLougheed KE, Bennett MH, Williams HD. An in vivo crosslinking system for identifying mycobacterial protein-protein interactions.. J Microbiol Methods 14 Jul 2014 (Journal article) Author URL J Microbiol Methods 14 Jul 2014  Author URL
  • Young NF, Ferguson BJ, Antoniadi I, Bennett MH, Beveridge CA, Turnbull CG. Conditional auxin response and differential cytokinin profiles in shoot branching mutants.. Plant Physiol 05 Jun 2014  Author URL   
  • Engl C, Waite CJ, McKenna JF, Bennett MH, Hamann T, Buck M. Chp8, a Diguanylate Cyclase from Pseudomonas syringae pv. Tomato DC3000, Suppresses the Pathogen-Associated Molecular Pattern Flagellin, Increases Extracellular Polysaccharides, and Promotes Plant Immune Evasion.. MBio 5(3) 2014  Author URL  

2013

  • Liebeke, M., Garcia-Perez, I., Anderson, C. J., Lawlor, A. J., Bennett, M. H., Morris, C. A.,Bundy, J. G. (2013). Earthworms Produce phytochelatins in Response to Arsenic.. PLoS One, 8(11), e81271. doi:10.1371/journal.pone.0081271
  • Schumacher, J., Behrends, V., Pan, Z., Brown, D. R., Heydenreich, F., Lewis, M. R., . . . Buck, M. (2013). Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC In Vivo.. MBio, 4(6). doi:10.1128/mBio.00881-13
  • Luang-In V, Narbad A, Nueno-Palop C, Mithen R, Bennett M, Rossiter JT .The metabolism of methylsulfinylalkyl- and methylthioalkyl-glucosinolates by a selection of human gut bacteria. MOL NUTR FOOD RES 30 Oct 2013 (Journal article) Author URL
  • Fones HN, Eyles CJ, Bennett MH, Smith JAC, Preston GM. Uncoupling of reactive oxygen species accumulation and defence signalling in the metal hyperaccumulator plant Noccaea caerulescens. NEW PHYTOLOGIST 199(4):916-924 01 Sep 2013 (Journal article) Author URL
  • Westwood JH, McCann L, Naish M, Dixon H, Murphy AM, Stancombe MA, Bennett MH, Powell G, Webb AAR, Carr JP. A viral RNA silencing suppressor interferes with abscisic acid-mediated signalling and induces drought tolerance in Arabidopsis thaliana. MOLECULAR PLANT PATHOLOGY 14(2):158-170 01 Feb 2013 (Journal article) Author URL
  • Williams KJ, Bennett MH, Barton GR, Jenkins VA, Robertson BD. Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation.. TUBERCULOSIS 93(2):198-206 01 Mar 2013 (Journal article) Author URL

2012

  • Hodge S, Ward JL, Beale MH, Bennett M, Mansfield JW, Powell G. Aphid-induced accumulation of trehalose in Arabidopsis thaliana is systemic and dependent upon aphid density.. PLANTA 237(4):1057-1064 01 Apr 2013 (Journal article) Author URL
  • Trauner A, Lougheed KEA, Bennett MH, Hingley-Wilson SM, Williams HD. The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria.. JOURNAL OF BIOLOGICAL CHEMISTRY 287(28):24053-24063 06 Jul 2012 (Journal article) Author URL
  • Pham J, Liu J, Bennett MH, Mansfield JW, Desikan R. Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial and fungal infection.. NEW PHYTOLOGIST 194(1):168-180 01 Apr 2012 (Journal article) Author URL

2011

  • Denness L, McKenna JF, Segonzac C, Wormit A, Madhou P, Bennett M, Mansfield J, Zipfel C, Hamann T. Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis.. PLANT PHYSIOLOGY 156(3):1364-1374 01 Jul 2011 (Journal article) Author URL
  • Trauner A, Bennett MH, Williams HD. Isolation of bacterial ribosomes with monolith chromatography.. PLOS ONE 6(2):6 pages Article number ARTN e16273 04 Feb 2011 (Journal article) Author URL

2010

  • Ward JL, Forcat S, Beckmann M, Bennett M, Miller SJ, Baker JM, Hawkins ND, Vermeer CP, Lu C, Lin W, et al.. The metabolic transition during disease following infection of Arabidopsis thaliana by Pseudomonas syringae pv. tomato. PLANT JOURNAL 63(3):443-457 01 Aug 2010 (Journal article) Author URL
  • Lewsey MG, Murphy AM, MacLean D, Dalchau N, Westwood JH, Macaulay K, Bennett MH, Moulin M, Hanke DE, Powell G, et al.. Disruption of Two Defensive Signaling Pathways by a Viral RNA Silencing Suppressor. MOLECULAR PLANT-MICROBE INTERACTIONS 23(7):835-845 01 Jul 2010 (Journal article) Author URL
  • Lewsey MG, Murphy AM, MacLean D, Dalchau N, Westwo od J, Macaulay K, Bennett MH, Moulin M, Hanke DE, Powell G, et al.. Disruption of the salicylate and jasmonate signaling pathways by the cucumber mosaic virus 2b RNA silencing suppressor. Annual Meeting of the American-Phytopathological-Society (APS), Charlotte, NC, 07 Aug 2010 - 11 Aug 2010. P HYTOPATHOLOGY. AMER PHYTOPATHOLOGICAL SOC. 100: S70-S70. 01 Jun 2010 (Conference) Author URL
  • Forcat S, Bennett M, Grant M, Mansfield JW. Rapid linkage of indole carboxylic acid to the plant cell wall identified as a component of basal defence in Arabidopsis against hrp mutant bacteria.. PHYTOCHEMISTRY 71(8-9):870-876 01 Jun 2010 (Journal article) Author URL
  • Rico A, Bennett MH, Forcat S, Huang WE, Preston GM. Agroinfiltration Reduces ABA Levels and Suppresses Pseudomonas syringae-Elicited Salicylic Acid Production in Nicotiana tabacum. PLOS ONE 5(1):12 pages Article number ARTN e8977 29 Jan 2010 (Journal article) Author URL
  • Truman WM, Bennett MH, Turnbull CGN, Grant MR. Arabidopsis auxin mutants are compromised in systemic acquired resistance and exhibit aberrant accumulation of various indolic compounds.. PLANT PHYSIOLOGY 152(3):1562-1573 01 Mar 2010 (Journal article) Author URL

2009

  • Zabala MDT, Bennett MH, Truman WH, Grant MR. Antagonism between salicylic and abscisic acid reflects early host-pathogen conflict and moulds plant defence responses. PLANT JOURNAL 59(3):375-386 01 Aug 2009 (Journal article) Author URL
  • Hamann T, Bennett M, Mansfield J, Somerville C. Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses.. PLANT JOURNAL 57(6):1015-1026 01 Mar 2009 (Journal article) Author URL

2008

  • Forcat S, Bennett MH, Mansfield JW, Grant MR. A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress.. PLANT METHODS 4:8 pages Article number ARTN 16 30 Jun 2008 (Journal article)Author URL

2007

  • Rossiter JT, Pickett JA, Bennett MH, Bones AM, Powell G, Cobb J. The synthesis and enzymic hydrolysis of (E)-2-[2,3-2H2]propenyl glucosinolate: confirmation of the rearrangement of the thiohydroximate moiety.. PHYTOCHEMISTRY 68(10):1384-1390 01 May 2007 (Journal article)Author URL
  • de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, Mansfield JW, Rodriguez Egea P, Bögre L, Grant M. Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease.. EMBO J 26(5):1434-1443 07 Mar 2007 (Journal article)Author URL
  • Truman W, Bennettt MH, Kubigsteltig I, Turnbull C, Grant M. Arabidopsis systemic immunity uses conserved defense signaling pathways and is mediated by jasmonates.. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104(3):1075-1080 16 Jan 2007 (Journal article) Author URL

2006

  • Jones AME, Thomas V, Bennett MH, Mansfield J, Grant M. Modifications to the Arabidopsis defense proteome occur prior to significant transcriptional change in response to inoculation with Pseudomonas syringae.. PLANT PHYSIOLOGY 142(4):1603-1620 01 Dec 2006 (Journal article)Author URL
  • Jones AME, Bennett MH, Mansfield JW, Grant M. Analysis of the defence phosphoproteome of Arabidopsis thaliana using differential mass tagging.. PROTEOMICS 6(14):4155-4165 01 Jul 2006 (Journal article) Author URL
  • Grant M, Truman B, Zabala MDT, Bennett M, Turnbull C. Towards a systems approach to plant defense responses. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY A-MOLECULAR & INTEGRATIVE PHYSIOLOGY 143(4):S137-S137 01 Apr 2006 (Journal article) Author URL

2005

  • Hoque ME, Mansfield JW, Bennett MH. Agrobacterium-mediated transformation of Indica rice genotypes: an assessment of factors affecting the transformation efficiency. PLANT CELL TISSUE AND ORGAN CULTURE 82(1):45-55 01 Jul 2005 (Journal article) Author URL

2003

  • Mert-Turk F, Bennett MH, Mansfield JW, Holub EB. Camalexin accumulation in Arabidopsis thaliana following abiotic elicitation or inoculation with virulent or avirulent Hyaloperonospora parasitica. PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY 62(3):137-145 01 Mar 2003 (Journal article) Author URL
  • Mert-Turk F, Bennett MH, Mansfield JW, Holub EB. Quantification of camalexin in several accessions of Arabidopsis thaliana following inductions with Peronospora parasitica and UV-B irradiation. PHYTOPARASITICA 31(1):81-89 01 Jan 2003 (Journal article) Author URL