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

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 Sym posium EU-Russia: Prospects for Cooperation in Biotechnology in the Seventh Fram ew ork Program m e

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

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

qv xB qv xB

B

v v

FTMS basics

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

Ultrahigh resolution

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

Ultrahigh resolution

Ultrahigh resolution

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

0.026 Da

Ultrahigh m ass precision

Exact mass [Da] Error [µDa]

1H

1.00782503214 0.00035

2H

2.01410177799 0.00036

3H

3.0160492675 0.0011

3He

3.01602930970 0.00086

4He

4.0026032497 0.0010

13C

13.0033548378 0.0010

12C

12.0000000000 0.0010

14C

14.0032419884 0.0040

14N

14.00307400524 0.00086

15N

15.00010889844 0.00092

16O

15.9949146221 0.0015

17O

16.999131501 0.22

18O

17.999160419 0.9

20Ne 19.9924401759

0.0020

23Na 22.989769675

0.23

28Si

27.9769265327 0.0020

31P

30.973761512 0.20

32S

31.972070690 0.12

34S

33.967866831 0.11

39K

38.963706861 0.3

40Ar 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

ERRORS C14H26NO3 C14H25NO3 + H+

Ultrahigh resolution

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

Bias elem ination by m ass precision

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

O/C atomic ratio H/C atomic ratio

5 ppm 2210 formulae calculated

Bias elem ination by m ass precision

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

O/C atomic ratio H/C atomic ratio

5 ppm 2 ppm 1012 formulae calculated

Bias elem ination by m ass precision

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

O/C atomic ratio H/C atomic ratio

2 ppm 1 ppm 743 formulae calculated

Bias elem ination by m ass precision

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

O/C atomic ratio H/C atomic ratio

1 ppm 0.5 ppm 639 formulae calculated

Fields of interest

Rhizosphere interactions

Bauer & Mathesius, Curr. Opin. Plant Biol., 2004, adapted

Bacteria Fungi Protozoa Plant

Quorum Sensing

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

Quorum Sensing

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

Finding: luminescent bacteria from marine symbioses regulate light production in dependance of their cell density. Explanation: changes in the

• bserved “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?

Signalling m olecules

N-Acylhom oserine Lactones

Environm ent and Health

Targeting rhizosphere bacteria

Frommberger et al., Anal. Bioanal. Chem., 2004, 378, 1014

?

LA-5 N-35

Retention Time [ min] Retention Time [ min]

Nano-LC/Ion Trap MS

AU 0.00 0.20 0.40 0.60 0.80 1.00 1.20 Minutes 0.20 0.40 0.60 0.80 1.00 1.20 1.40

UPLC

Fekete et al., Electrophoresis, submitted.

C4 C6 C7 C8 C10 C12 C14

Target analysis

294.14667 294.16765 294.10 294.12 294.14 294.16 294.18 294.20 294.22 m/z 0.0 0.5 1.0 1.5 6 x10 Intens.

[C14H25NO4+Na]+ Theory: 294,16757 amu Exp.: 297,16766 amu Error: -0,291 ppm

C4H8NO2 Δ 1.230 ppm

Confirmation by FTICR-MS

Com petition in the rhizosphere

Cooperation at GSF: M. Schloter, F. Häsler

Actinobacterium (A) Plant pathogen (P) Mechanisms of PGPR activity

Cocultivation in liquid m edium

251.12544 252.97384 255.23194 258.25094 261.13090 263.23694 265.25265 271.26323 273.16971 277.21386 279.22953 282.06453 285.27885 286.96956 288.28978 292.82347 295.19453 298.04211 301.14108 302.95624 305.24513 308.03161 311.25819 313.27378 316.25640 322.00866 324.00559 326.00438 327.15676 329.23232 336.06287 337.23498 343.13065 347.24281 349.18332 353.20890 355.28434 359.27928 363.11145 365.15723 367.33597 369.35160 381.26108 383.20403 385.34647

1 2 260 280 300 320 340 360 380 m/z

251.12545 252.97382 255.23193 261.13094 263.23705 265.25264 268.10410 271.26322 273.99580 277.21388 279.15914 282.06453 283.26321 285.27885 286.96955 288.96666 295.19450 298.04211 301.14107 302.95627 305.24513 308.03163 310.02982 313.27378 316.25639 323.14656 326.00431 327.15673 329.23231 336.06287 337.23496 341.19991 343.13045 347.24283 353.09171 359.27926 361.22212 363.11148 369.35159 371.31562 376.00751 381.26106 383.20402 385.34653

1 2

251.12548 255.23198 257.24762 261.13098 265.16468 269.24766 271.26328 275.11026 277.21394 279.15918 285.27892 286.96962 290.07488 294.13373 298.04217 301.14115 305.15482 309.27895 311.29457 313.27386 315.25314 320.12603 322.16262 327.20147 329.23240 334.12627 337.23509 339.13006 341.25488 343.08403 348.08037 353.21712 355.27049 359.16035 363.16266 366.12255 369.35173 371.22767 373.17558 376.19122 384.67516

1 2

251.12548 252.97389 255.23198 257.24761 261.13097 264.15953 266.11363 271.26329 275.06396 277.17768 279.15918 285.27891 286.96962 288.96674 294.13371 296.13549 298.04217 301.14115 305.15510 308.08783 312.11908 315.25313 320.12599 322.16260 327.15686 329.23239 334.12626 337.23507 339.13006 341.25487 343.08404 348.08035 353.20905 355.27047 359.16030 362.13478 366.12255 368.09529 369.35171 371.31573 373.17555 376.19118 380.13549 383.20394 384.67510

1 2

Intens.

8

x10

251.12544 252.97384 255.23194 258.25094 261.13090 263.23694 265.25265 271.26323 273.16971 277.21386 279.22953 282.06453 285.27885 286.96956 288.28978 292.82347 295.19453 298.04211 301.14108 302.95624 305.24513 308.03161 311.25819 313.27378 316.25640 322.00866 324.00559 326.00438 327.15676 329.23232 336.06287 337.23498 343.13065 347.24281 349.18332 353.20890 355.28434 359.27928 363.11145 365.15723 367.33597 369.35160 381.26108 383.20403 385.34647

1 2 260 280 300 320 340 360 380 m/z

251.12545 252.97382 255.23193 261.13094 263.23705 265.25264 268.10410 271.26322 273.99580 277.21388 279.15914 282.06453 283.26321 285.27885 286.96955 288.96666 295.19450 298.04211 301.14107 302.95627 305.24513 308.03163 310.02982 313.27378 316.25639 323.14656 326.00431 327.15673 329.23231 336.06287 337.23496 341.19991 343.13045 347.24283 353.09171 359.27926 361.22212 363.11148 369.35159 371.31562 376.00751 381.26106 383.20402 385.34653

1 2

251.12548 255.23198 257.24762 261.13098 265.16468 269.24766 271.26328 275.11026 277.21394 279.15918 285.27892 286.96962 290.07488 294.13373 298.04217 301.14115 305.15482 309.27895 311.29457 313.27386 315.25314 320.12603 322.16262 327.20147 329.23240 334.12627 337.23509 339.13006 341.25488 343.08403 348.08037 353.21712 355.27049 359.16035 363.16266 366.12255 369.35173 371.22767 373.17558 376.19122 384.67516

1 2

251.12548 252.97389 255.23198 257.24761 261.13097 264.15953 266.11363 271.26329 275.06396 277.17768 279.15918 285.27891 286.96962 288.96674 294.13371 296.13549 298.04217 301.14115 305.15510 308.08783 312.11908 315.25313 320.12599 322.16260 327.15686 329.23239 334.12626 337.23507 339.13006 341.25487 343.08404 348.08035 353.20905 355.27047 359.16030 362.13478 366.12255 368.09529 369.35171 371.31573 373.17555 376.19118 380.13549 383.20394 384.67510

1 2

Intens.

8

x10

Media Blank Pathogen Actinobacterium Actinobacterium + Pathogen

Cocultivation in liquid m edium

251.12544 252.97384 255.23194 258.25094 261.13090 263.23694 265.25265 271.26323 273.16971 277.21386 279.22953 282.06453 285.27885 286.96956 288.28978 292.82347 295.19453 298.04211 301.14108 302.95624 305.24513 308.03161 311.25819 313.27378 316.25640 322.00866 324.00559 326.00438 327.15676 329.23232 336.06287 337.23498 343.13065 347.24281 349.18332 353.20890 355.28434 359.27928 363.11145 365.15723 367.33597 369.35160 381.26108 383.20403 385.34647

1 2 260 280 300 320 340 360 380 m/z

251.12545 252.97382 255.23193 261.13094 263.23705 265.25264 268.10410 271.26322 273.99580 277.21388 279.15914 282.06453 283.26321 285.27885 286.96955 288.96666 295.19450 298.04211 301.14107 302.95627 305.24513 308.03163 310.02982 313.27378 316.25639 323.14656 326.00431 327.15673 329.23231 336.06287 337.23496 341.19991 343.13045 347.24283 353.09171 359.27926 361.22212 363.11148 369.35159 371.31562 376.00751 381.26106 383.20402 385.34653

1 2

251.12548 255.23198 257.24762 261.13098 265.16468 269.24766 271.26328 275.11026 277.21394 279.15918 285.27892 286.96962 290.07488 294.13373 298.04217 301.14115 305.15482 309.27895 311.29457 313.27386 315.25314 320.12603 322.16262 327.20147 329.23240 334.12627 337.23509 339.13006 341.25488 343.08403 348.08037 353.21712 355.27049 359.16035 363.16266 366.12255 369.35173 371.22767 373.17558 376.19122 384.67516

1 2

251.12548 252.97389 255.23198 257.24761 261.13097 264.15953 266.11363 271.26329 275.06396 277.17768 279.15918 285.27891 286.96962 288.96674 294.13371 296.13549 298.04217 301.14115 305.15510 308.08783 312.11908 315.25313 320.12599 322.16260 327.15686 329.23239 334.12626 337.23507 339.13006 341.25487 343.08404 348.08035 353.20905 355.27047 359.16030 362.13478 366.12255 368.09529 369.35171 371.31573 373.17555 376.19118 380.13549 383.20394 384.67510

1 2

Intens.

8

x10

251.12544 252.97384 255.23194 258.25094 261.13090 263.23694 265.25265 271.26323 273.16971 277.21386 279.22953 282.06453 285.27885 286.96956 288.28978 292.82347 295.19453 298.04211 301.14108 302.95624 305.24513 308.03161 311.25819 313.27378 316.25640 322.00866 324.00559 326.00438 327.15676 329.23232 336.06287 337.23498 343.13065 347.24281 349.18332 353.20890 355.28434 359.27928 363.11145 365.15723 367.33597 369.35160 381.26108 383.20403 385.34647

1 2 260 280 300 320 340 360 380 m/z

251.12545 252.97382 255.23193 261.13094 263.23705 265.25264 268.10410 271.26322 273.99580 277.21388 279.15914 282.06453 283.26321 285.27885 286.96955 288.96666 295.19450 298.04211 301.14107 302.95627 305.24513 308.03163 310.02982 313.27378 316.25639 323.14656 326.00431 327.15673 329.23231 336.06287 337.23496 341.19991 343.13045 347.24283 353.09171 359.27926 361.22212 363.11148 369.35159 371.31562 376.00751 381.26106 383.20402 385.34653

1 2

251.12548 255.23198 257.24762 261.13098 265.16468 269.24766 271.26328 275.11026 277.21394 279.15918 285.27892 286.96962 290.07488 294.13373 298.04217 301.14115 305.15482 309.27895 311.29457 313.27386 315.25314 320.12603 322.16262 327.20147 329.23240 334.12627 337.23509 339.13006 341.25488 343.08403 348.08037 353.21712 355.27049 359.16035 363.16266 366.12255 369.35173 371.22767 373.17558 376.19122 384.67516

1 2

251.12548 252.97389 255.23198 257.24761 261.13097 264.15953 266.11363 271.26329 275.06396 277.17768 279.15918 285.27891 286.96962 288.96674 294.13371 296.13549 298.04217 301.14115 305.15510 308.08783 312.11908 315.25313 320.12599 322.16260 327.15686 329.23239 334.12626 337.23507 339.13006 341.25487 343.08404 348.08035 353.20905 355.27047 359.16030 362.13478 366.12255 368.09529 369.35171 371.31573 373.17555 376.19118 380.13549 383.20394 384.67510

1 2

Intens.

8

x10

Media Blank Pathogen Actinobacterium Actinobacterium + Pathogen

?

Analytical approach

Substance identification by FTICR-MS (hopefully!) Activity assays of SPE fractions (V8 agar, together with P) Chromatographic fractionation of supernatant (C18 SPE) Activity assay of supernatant (V8 agar, together with P) Cultivation of Actinomycete in synthetic medium (reduces complexity)

Chrom atographic fractionation

Cooperation at GSF: M. Schloter, F. Häsler

Elution with 30% MeOH Elution with 40% MeOH Elution with 50% MeOH

I dentification by FTMS

Cooperation at GSF: M. Schloter, F. Häsler

214.08956 236.07152 252.97370 282.16995 302.87768 322.00860 343.08394 383.20407 399.17802 432.04909 450.00352

1 2 3 4 8 x10 Intens.

214.08953 246.14878 264.15935 282.16992 304.15191 323.14651 344.09177 362.02657 373.03731 481.13152 499.14218

1 2 3 4 5 8 x10 200 250 300 350 400 450 500 550 600 m/z 200 250 300 350 400 450 500 550 600 m/z

214.08960 252.97373 315.25300 341.25471 361.22209 383.20405 408.08407 444.17932 484.17294 518.19170 596.31937 626.33001

1 2 3 4 5 8 x10 200 250 300 350 400 450 500 550 600 m/z

30% 40% 50%

214.08953 246.14878 264.15935 282.16992 304.15191 323.14651 344.09177 362.02657 373.03731 481.13152 499.14218

1 2 3 4 5 8 x10

I dentification by FTMS

Cooperation at GSF: M. Schloter, F. Häsler

40%

282.16995

2 4

283.17330

2 4

8

• Intens. x10

282.16998

2 4 282.0 282.2 282.4 282.6 282.8 283.0 283.2 m/z

I dentification by FTMS

Cooperation at GSF: M. Schloter, F. Häsler

40%

282.16992

NH O O OH H O H3C H3C

C15H24NO4 (error 0.213 ppm) Cycloheximide

Constitutive or induced?

251.12544 252.97384 255.23194 258.25094 261.13090 263.23694 265.25265 271.26323 273.16971 277.21386 279.22953 282.06453 285.27885 286.96956 288.28978 292.82347 295.19453 298.04211 301.14108 302.95624 305.24513 308.03161 311.25819 313.27378 316.25640 322.00866 324.00559 326.00438 327.15676 329.23232 336.06287 337.23498 343.13065 347.24281 349.18332 353.20890 355.28434 359.27928 363.11145 365.15723 367.33597 369.35160 381.26108 383.20403 385.34647

1 2 260 280 300 320 340 360 380 m/z

251.12545 252.97382 255.23193 261.13094 263.23705 265.25264 268.10410 271.26322 273.99580 277.21388 279.15914 282.06453 283.26321 285.27885 286.96955 288.96666 295.19450 298.04211 301.14107 302.95627 305.24513 308.03163 310.02982 313.27378 316.25639 323.14656 326.00431 327.15673 329.23231 336.06287 337.23496 341.19991 343.13045 347.24283 353.09171 359.27926 361.22212 363.11148 369.35159 371.31562 376.00751 381.26106 383.20402 385.34653

1 2

251.12548 255.23198 257.24762 261.13098 265.16468 269.24766 271.26328 275.11026 277.21394 279.15918 285.27892 286.96962 290.07488 294.13373 298.04217 301.14115 305.15482 309.27895 311.29457 313.27386 315.25314 320.12603 322.16262 327.20147 329.23240 334.12627 337.23509 339.13006 341.25488 343.08403 348.08037 353.21712 355.27049 359.16035 363.16266 366.12255 369.35173 371.22767 373.17558 376.19122 384.67516

1 2

251.12548 252.97389 255.23198 257.24761 261.13097 264.15953 266.11363 271.26329 275.06396 277.17768 279.15918 285.27891 286.96962 288.96674 294.13371 296.13549 298.04217 301.14115 305.15510 308.08783 312.11908 315.25313 320.12599 322.16260 327.15686 329.23239 334.12626 337.23507 339.13006 341.25487 343.08404 348.08035 353.20905 355.27047 359.16030 362.13478 366.12255 368.09529 369.35171 371.31573 373.17555 376.19118 380.13549 383.20394 384.67510

1 2

Intens.

8

x10

251.12544 252.97384 255.23194 258.25094 261.13090 263.23694 265.25265 271.26323 273.16971 277.21386 279.22953 282.06453 285.27885 286.96956 288.28978 292.82347 295.19453 298.04211 301.14108 302.95624 305.24513 308.03161 311.25819 313.27378 316.25640 322.00866 324.00559 326.00438 327.15676 329.23232 336.06287 337.23498 343.13065 347.24281 349.18332 353.20890 355.28434 359.27928 363.11145 365.15723 367.33597 369.35160 381.26108 383.20403 385.34647

1 2 260 280 300 320 340 360 380 m/z

251.12545 252.97382 255.23193 261.13094 263.23705 265.25264 268.10410 271.26322 273.99580 277.21388 279.15914 282.06453 283.26321 285.27885 286.96955 288.96666 295.19450 298.04211 301.14107 302.95627 305.24513 308.03163 310.02982 313.27378 316.25639 323.14656 326.00431 327.15673 329.23231 336.06287 337.23496 341.19991 343.13045 347.24283 353.09171 359.27926 361.22212 363.11148 369.35159 371.31562 376.00751 381.26106 383.20402 385.34653

1 2

251.12548 255.23198 257.24762 261.13098 265.16468 269.24766 271.26328 275.11026 277.21394 279.15918 285.27892 286.96962 290.07488 294.13373 298.04217 301.14115 305.15482 309.27895 311.29457 313.27386 315.25314 320.12603 322.16262 327.20147 329.23240 334.12627 337.23509 339.13006 341.25488 343.08403 348.08037 353.21712 355.27049 359.16035 363.16266 366.12255 369.35173 371.22767 373.17558 376.19122 384.67516

1 2

251.12548 252.97389 255.23198 257.24761 261.13097 264.15953 266.11363 271.26329 275.06396 277.17768 279.15918 285.27891 286.96962 288.96674 294.13371 296.13549 298.04217 301.14115 305.15510 308.08783 312.11908 315.25313 320.12599 322.16260 327.15686 329.23239 334.12626 337.23507 339.13006 341.25487 343.08404 348.08035 353.20905 355.27047 359.16030 362.13478 366.12255 368.09529 369.35171 371.31573 373.17555 376.19118 380.13549 383.20394 384.67510

1 2

Intens.

8

x10

Media Blank P A A + P

Cycloheximide x2

Tobacco m etabolites

Tobacco m etabolites

Tobacco m etabolites

Tobacco m etabolites

Much m ore ( … and still not com plete …)

Peptide biomarkers for cancer and kidney diseases

• H. Mischak, P. Zürbig, Mosaiques AG, Hannover

Chemical Taxonomy of bacteria ecotypes

• R. Rossello-Mora, IMEDEA, Mallorca

Arabidopsis mutant analysis

• T. Schäffner, B. Messner, BIOP, GSF

Tobacco metabolomics

• K. Wieland, U. Schurr, FZ Jülich

Chemical Ecology of caterpillars

• W. Boland H. Meischak, MPI of Chemical Ecology, Jena

UV/ozone induced metabolites of different plants

• W. Heller, BIOP, GSF

Different questions in groundwater ecology

• U. Kunapuli, R. Meckenstock, IGOE, GSF

Geochemistry, NOM, DOM, DOC, Aerosols, Wine … Philippe Schmitt-Kopplin

Much m ore here …

Thank you!

Большое спасибо!