An Analytical Platform for Environmental, Plant and Microbial - PowerPoint PPT Presentation

Sym posium EU-Russia: Prospects for Cooperation in Biotechnology in the Seventh Fram ew ork Program m e An Analytical Platform for Environmental, Plant and Microbial Metabolomics Based on Ultrahigh Resolution Mass Spectrometry (FTMS) Moritz Frommberger, Norbert Hertkorn, Philippe Schmitt-Kopplin GSF Research Center for Environment and Health Institute of Ecological Chemistry Molecular BioGeoAnalysis Munich, Germany

Analytical platform

Contents FTICR Principles Analytical approaches Analysis of interactions in the Rhizosphere: N -Acylhomoserine lactones and Quorum Sensing Mechanism of PGPR activity in soil Actinobacteria Other Projects

FTMS basics B v v q v x B q v x B Marshall et al., Mass Spectrom. Rev. , 1998, 17 , 1

FTMS basics Marshall et al., Mass Spectrom. Rev. , 1998, 17 , 1

Ultrahigh resolution Intens. x10 0.0 0.5 1.0 1.5 2.0 2.5 7 266.98864 276.33481 300 297.09817 303.58445 311.11373 327.10868 339.10869 350 353.12435 369.11924 381.11929 400 397.11420 411.12987 425.14559 439.12482 450 453.14054 469.13552 481.13549 495.15117 500 511.14606 525.16173 539.17732 550 553.15691 567.17281 583.16784 600 597.18287 bkoch_000003.d: -MS 611.19870 621.18274 637.17863 650 m/z

Ultrahigh resolution

Ultrahigh resolution Intens. Intens. 282.16373 7 7 x10 x10 282.06444 1.0 1.0 0.8 0.8 0.6 0.6 282.03833 0.4 0.4 0.026 Da 282.13765 281.99585 0.2 0.2 282.07217 282.22163 282.27904 0.0 0.0 281.7 281.7 281.8 281.8 281.9 281.9 282.0 282.0 282.1 282.1 282.2 282.2 282.3 282.3 282.4 282.4 282.5 m/z 282.5 m/z

Ultrahigh m ass precision Exact mass [Da] Error [µDa] 1 H 1.00782503214 0.00035 2 H 2.01410177799 0.00036 3 H 3.0160492675 0.0011 3 He 3.01602930970 0.00086 4 He 4.0026032497 0.0010 13 C 13.0033548378 0.0010 12 C 12.0000000000 0.0010 14 C 14.0032419884 0.0040 14 N 14.00307400524 0.00086 15 N 15.00010889844 0.00092 16 O 15.9949146221 0.0015 17 O 16.999131501 0.22 18 O 17.999160419 0.9 20 Ne 19.9924401759 0.0020 23 Na 22.989769675 0.23 28 Si 27.9769265327 0.0020 31 P 30.973761512 0.20 32 S 31.972070690 0.12 34 S 33.967866831 0.11 39 K 38.963706861 0.3 40 Ar 39.9623831232 0.0030 Audi, G. & Wapstra, A. H. Nucl. Phys. A, 1995, 595 , 409

Ultrahigh m ass precision Only limited number of elemental combinations fits into an exact molecular mass C 14 H 26 NO 3 ERRORS C 14 H 25 NO 3 + H +

Ultrahigh resolution Intens. x10 0.0 0.5 1.0 1.5 2.0 2.5 7 266.98864 276.33481 300 297.09817 303.58445 311.11373 327.10868 339.10869 350 353.12435 369.11924 381.11929 400 397.11420 411.12987 425.14559 439.12482 450 453.14054 469.13552 481.13549 495.15117 500 511.14606 525.16173 539.17732 550 553.15691 567.17281 583.16784 600 597.18287 bkoch_000003.d: -MS 611.19870 621.18274 637.17863 650 m/z

Bias elem ination by m ass precision 5 ppm 2 1.8 1.6 1.4 H/C atomic ratio 1.2 1 0.8 0.6 0.4 2210 formulae calculated 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O/C atomic ratio

Bias elem ination by m ass precision 2 5 ppm 2 ppm 1.8 1.6 1.4 H/C atomic ratio 1.2 1 0.8 0.6 0.4 1012 formulae calculated 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O/C atomic ratio

Bias elem ination by m ass precision 2 2 ppm 1 ppm 1.8 1.6 1.4 H/C atomic ratio 1.2 1 0.8 0.6 0.4 743 formulae calculated 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O/C atomic ratio

Bias elem ination by m ass precision 2 1 ppm 0.5 ppm 1.8 1.6 1.4 H/C atomic ratio 1.2 1 0.8 0.6 0.4 639 formulae calculated 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 O/C atomic ratio

Fields of interest

Rhizosphere interactions Bacteria Plant Fungi Protozoa Bauer & Mathesius, Curr. Opin. Plant Biol. , 2004, adapted

Quorum Sensing Fuqua & Greenberg, Nat. Rev. Mol. Cell Biol., 2002, 3 , 4111

Quorum Sensing Finding: luminescent bacteria from marine symbioses regulate light production in dependance of their cell density. Explanation: changes in the observed “physiological behavior” are a consequence of production, accumulation of, and reaction to, specific signalling molecules. Conclusion: bacteria are able to actively communicate, not only to react to. � First step to multicellularity? Fuqua & Greenberg, Nat. Rev. Mol. Cell Biol., 2002, 3 , 4111

Signalling m olecules

N-Acylhom oserine Lactones

Environm ent and Health

Targeting rhizosphere bacteria Nano-LC/Ion Trap MS LA-5 N-35 ? Retention Time [ min] Retention Time [ min] Frommberger et al., Anal. Bioanal. Chem ., 2004, 378 , 1014

UPLC C7 1.20 C6 C8 1.00 C10 C12 C4 0.80 C14 AU 0.60 0.40 0.20 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 Minutes Fekete et al., Electrophoresis , submitted.

Target analysis Intens. 6 Confirmation by FTICR-MS x10 294.16765 294.14667 1.5 [C 14 H 25 NO 4 +Na] + Theory: 294,16757 amu Exp.: 297,16766 amu Error: -0,291 ppm 1.0 Intens. 5 x10 102.054829 5 4 C 4 H 8 NO 2 0.5 Δ 1.230 ppm 3 2 102.038896 102.066241 102.032167 102.010107 1 102.048621 102.023096 0.0 0 294.10 294.12 294.14 294.16 294.18 294.20 294.22 m/z 102.00 102.01 102.02 102.03 102.04 102.05 102.06 102.07 102.08 m/z +MS

Com petition in the rhizosphere Mechanisms of PGPR activity Plant pathogen (P) Actinobacterium (A) Cooperation at GSF: M. Schloter, F. Häsler