description of techniques used in metabolite profiling of bryophytes

1. Department of Botany, Panjab University, Chandigarh
Presented by:
Unnati
MSc II(4th sem)
Date: April 5, 2019
TECHNIQUES USED IN METABOLITE PROFILING OF
BRYOPHYTES

2. Outline of Presentation
 Introduction
 Definition
 Terminology
 Techniques of metabolite
profiling
o GC-MS
o LC-MS
o FTICR-MS
o CE-MS
o NMR Spectroscopy
 Future Recommendation
 Conclusion
 Acknowledgement
 References

3. Bryophytes are the most primitive, non-vascular, first terrestrial plants which pioneered the
process of conquest on the land. It is the second largest group in the plant kingdom after
angiosperms. Bryophytes can be found in any habitat globally where photosynthesis is possible.
Bryophytes are divided into three groups:-
1. Mosses
2. Liverworts
3. Hornworts
An important direction in bryophyte studies is analysis of their biologically active substances,
particularly the secondary metabolites. Studies on biologically active compounds in bryophytes
are rapidly growing, resulting in identification of a large number of specific substances with high
biological activity.
Introduction/Brief History

4. Metabolite Profiling is the measurement in biological systems of the
complement of low-molecular-weight metabolites and their intermediates
that reflects the dynamic response to genetic modification and
physiological, pathophysiological and developmental stimuli.
What is Metabolite Profiling?

5. METABOLITE
FINGERPRINTING
METABOLITE
PROFILING
METABOLOMICS
It is the application of a broad
analytic technology to discover
some big differences between two
samples.
For e.g. two different genotypes
It is the measurement of hundreds
or potentially thousands of
metabolites.
It requires a streamlined pipeline
for extraction, separation and
analysis so that large number of
metabolites can be measured in a
quantitative manner.
It is the measurement of all
metabolites in a given system.
TERMINOLOGY

6.  Gas chromatography coupled to Mass Spectrometry (GC-MS)
 Liquid chromatography coupled to Mass Spectrometry (LC-MS)
 Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry
(FTICR-MS)
 Capillary Electrophoresis coupled to Mass Spectrometry (CE-MS)
 Nuclear Magnetic Resonance spectroscopy (NMR)
Techniques of Metabolite Profiling

7. GC-MS is an integrated composite analysis Instrument combining GC which is excellent in its
ability for separation with mass spectrometry which is ideal in identification and elucidate
structure of separated component.
Gas
Chromatography
It separates
components of
sample.
Interface
Combines both techniques by
removing pressure
incompatibility problem
between GC and MS
Mass spectrometry
Ionise eluted
component and
separate, Identify it
according to its mass
to charge ratio.
The use of mass spectrometer as the detector in gas chromatography was developed during
1959 by Roland Gohlke and Fred McLafferty.
Gas Chromatography coupled to Mass Spectrometry (GC-MS)

8. https://image.slidesharecdn.com/gc-msgood-130615222234-
phpapp01/95/gas-chromatography-and-mass-spectrometry-
gcms-by-pravisankar-3-638.jpg?cb=1371335021
https://image.slidesharecdn.com/gc-msgood-130615222234-phpapp01/95/gas-
chromatography-and-mass-spectrometry-gcms-by-pravisankar-17-
638.jpg?cb=1371335021
GC-MS

9. • To analyze small molecule metabolite identifications in the diabetic versus non diabetic urine
samples.
• Analysis of anabolic steroids in biological materials.
• Determination of furans in food beverages.
• The routine analysis of substances present in minute quantities.
• Identification of noxious and toxic compounds and their quantitation in emergency cases.
• Environmental monitoring and clean up.
Applications of GC-MS

10. GC-MS technique is used to study
metabolite profiling in moss Physcomitrella
patens.
A report on metabolite profiling of two
major P. patens tissues, filamentous
protonema and leafy gametophores, from
different culture conditions. A total of 96
compounds were detected, 21 of them as yet
unknown in public databases.
Physcomitrella patens
https://upload.wikimedia.org/wikipedia/commons/d/dd/Physcomitrella.jpg
EXAMPLE

11. • LC-MS is an analytical chemistry technique that combines the physical separation
capabilities of liquid chromatography with the mass analysis capabilities of mass
spectrometry.
Liquid
Chromatography Ionization Mass Analyzer
Detector/
Data collection
• LC-MS is a very powerful technique used for many application which has very high
sensitivity and specificity.
• It’s application is oriented towards the specific detection and potential identification of
chemicals in the presence of other chemicals (in a complex mixture).
Liquid Chromatography coupled to Mass Spectrometry (LC-
MS)

12. https://image.slidesharecdn.com/naveenjain-140804031638-
phpapp01/95/lcms-insturmentation-applications-20-
638.jpg?cb=1407122703
https://www.researchgate.net/profile/Mostafa_Abdelrahman10/publication/3179866
85/figure/fig2/AS:667887098396674@1536248101153/Schematic-representation-
of-the-liquid-chromatography-tandem-mass-spectrometry-LC-MS-MS.jpg
LC-MS

13.  Today toxicologists are faced with expectation of finding drugs and toxins in a variety of
materials, with smaller sample volume and at low concentration.
 Molecular weight determination.
 Structural determination e.g. structural determination of ginsenoside.
 Pharmaceutical application e.g. identification of bile acid metabolites.
 Biochemical application e.g. rapid protein identification.
 Environmental application e.g. detection of phenyl urea herbicides, detection of low level of
carbaryl in food.
Applications of LC-MS

14. LC-MS technique is used in Marchantia polymorpha, Plagiomnium undulatum, Polytrichum strictum, Grimmia
pulvinata to obtain metabolite profiles.
Marchantia polymorpha
https://upload.wikimedia.org/wikipedia/commons/thumb
/0/0a/Moos_5772.jpg/800px-Moos_5772.jpg
Polytrichum strictum
https://upload.wikimedia.org/wikipedia/commons/thumb/d/d2/Polytrichum_strictum_%28a%2C_150144
-481741%29_8419.JPG/1024px-Polytrichum_strictum_%28a%2C_150144-481741%29_8419.JPG
Example

15. Fourier Transform Ion Cyclotron Mass Spectrometry is a type of mass analyzer (or mass
spectrometer) for determining the mass-to-charge ratio of ions based on the cyclotron frequency
of the ions in a fixed magnetic field.
FT-ICR is the highest performance mass spectrometry
technique available, offering unrivalled resolution and
mass accuracy.
FTICR- MS is developed by Alan G. Marshall and
Melvin B. Comisarow in 1974 at the University of
British Columbia.
Fourier Transform Ion Cyclotron Resonance Mass Spectrome
try (FTICR-MS)
https://upload.wikimedia.org/wikipedia/commons/e/e6/LTQ-FTICR.jpg

16. http://www.mslab.ulg.ac.be/wp-content/uploads/2014/02/ft020.jpg http://www.mslab.ulg.ac.be/wp-content/uploads/2014/02/scheme-
ftms.gif
FTICR-MS

17.  One of the biggest advantage of FTICR-MS is its unparalleled mass resolution. Mass
measurements can be made on highly complex chemical or biological samples, without the
need of any separation method, such as gas or liquid chromatography.
 Also, the mass determination can be made to a minimum of 3 significant figures past the
decimal point by performing accurate mass measurements using an internal calibration.
 With this kind of accuracy, it is possible to determine the identity of unknowns based simply
on mass.
 Another unique feature of FTICR-MS is the control of stored ions within ion trap.
Applications of FTICR-MS

18. FTICR-MS technique is used in Physcomitrella patens. It is taken as model organism for the study of metabolite profiling in
bryophytes.
Physcomitrella patens
https://upload.wikimedia.org/wikipedia/commons/d/dd/Physcomitrella.jpg
Example

19. Capillary electrophoresis–mass spectrometry (CE-MS) is an analytical chemistry technique formed by the
combination of the liquid separation process of capillary electrophoresis with mass spectrometry. CE-MS combines
advantages of both CE and MS to provide high separation efficiency and molecular mass information in a single
analysis.
The original interface between capillary zone electrophoresis and
mass spectrometry was developed in 1987 by Richard D.
Smith and coworkers at Pacific Northwest National Laboratory,
and who also later were involved in development of interfaces
with other CE variants, including capillary isotachophoresis and
capillary isoelectric focusing.
https://upload.wikimedia.org/wikipedia/commons/thumb/6/64/S
chematic_Diagram_of_CE-ESI-MS_system.jpg/400px-
Schematic_Diagram_of_CE-ESI-MS_system.jpg
Capillary Electrophoresis coupled to Mass Spectrometry
(CE-MS)

20.  CE-MS ability to separate analytes present in extremely low concentration with high efficiency
at high speed has made it applicable in all fields of science.
 CE-MS has been used for bioanalytical, pharmaceuticals, environmental and forensic
application.
 The major application of CE-MS has been for biological studies, mostly for protein and
peptide analysis. Along with that, it is used often for routine analysis of pharmaceutical drugs.
 There are number of studies reporting characterization of mixtures of peptides and proteins.
 CE-MS can be used for routine clinical checkup. Body fluids like blood and urine have been
analyzed with CE-MS to identify biomarkers for renal diseases and cancer.
Applications of CE-MS

21. CE-MS technique is used to study the level of antioxidant compound Glutathione in moss from industrial areas. E.g.
Pseudoscleropodium purum (terrestrial moss).
Pseudoscleropodium purum
https://upload.wikimedia.org/wikipedia/commons/c/cd/Scleropodium
_purum0.jpg
Example

22.  Nuclear magnetic resonance spectroscopy, most commonly known as NMR
spectroscopy or magnetic resonance spectroscopy (MRS), is a spectroscopic technique which
is based on the absorption of electromagnetic radiation in the radio frequency range 4 to 900
MHz by nuclei of the atoms.
 NMR spectroscopy is the definitive method to identify monomolecular organic compounds.
 Biochemists use NMR to identify proteins and other complex molecules. Besides identification,
NMR spectroscopy provides detailed information about the structure, dynamics, reaction state,
and chemical environment of molecules.
 The most common types of NMR are proton and carbon-13 NMR spectroscopy, but it is
applicable to any kind of sample that contains nuclei possessing spin.
Nuclear Magnetic Resonance Spectroscopy (NMR)

23. NMR Instrumentation

24. • Solution structure The only method for atomic-resolution structure determination of biomacromolecules in aqueous solutions
under near physiological conditions or membrane mimeric environments.
• Molecular dynamics The most powerful technique for quantifying motional properties of biomacromolecules.
• Protein folding The most powerful tool for determining the residual structures of unfolded proteins and the structures of
folding intermediates.
• Ionization state The most powerful tool for determining the chemical properties of functional groups in biomacromolecules,
such as the ionization states of ionizable groups at the active sites of enzymes.
• Weak intermolecular interactions Allowing weak functional interactions between macrobiomolecules (e.g., those with
dissociation constants in the micromolar to millimolar range) to be studied, which is not possible with other technologies.
• Protein hydration A power tool for the detection of interior water and its interaction with biomacromolecules.
• Hydrogen bonding A unique technique for the direct detection of hydrogen bonding interactions.
• Drug screening and design Particularly useful for identifying drug leads and determining the conformations of the
compounds bound to enzymes, receptors, and other proteins.
• Native membrane protein Solid state NMR has the potential for determining atomic-resolution structures of domains of
membrane proteins in their native membrane environments, including those with bound ligands.
• Metabolite analysis A very powerful technology for metabolite analysis.
• Chemical analysis A matured technique for chemical identification and conformational analysis of chemicals whether
synthetic or natural.
• Material science A powerful tool in the research of polymer chemistry and physics.
Applications of NMR

25. NMR Spectroscopy is used to study biochemical analysis of different bryophytes.eg.
Leucobryum sanctum, Himantocladium cyclophyllum , Racomitrium lanuginosum etc.
Racomitrium lanuginosum
https://upload.wikimedia.org/wikipedia/commons/thumb/6/6c/Racomitr
ium_lanuginosum_%28c%2C_141022-472335%29_0960.JPG/800px-
Racomitrium_lanuginosum_%28c%2C_141022-472335%29_0960.JPG
Example

26. The separation of complex secondary metabolome mixtures is still quite challenging, and there
exists a need for greater differentiation and resolution in metabolomics approaches at both the
technical and biological levels. Efforts are amplifying the biological context of our metabolic
profiling efforts. This will continue to push forward this important area of research for the
advancement of plant productivity and for the improvement of human and animal nutrition and
health.
Future Recommendation

27. Measurements of metabolites provide basic information about biological
responses to physiological or environmental changes. Metabolite
profiling allows a shift from hypothesis- driven research to the analysis
of system-wide responses, especially when it is integrated with other
profiling technologies. This metabolite profiling can be used as a tool for
pharmaceutical industry.
Conclusion

28.  Gohlke, Roland S. and McLafferty, Fred W: Early gas chromatography/mass spectrometry. Journal of the
American Society for Mass Spectrometry 1993, 4 (5): 367–371
 Joachim Kopka, Alisdair Fernie, Wolfram Weckwerth, Yves Gibon and Mark Stitt: Metabolite profiling in plant
biology:platforms and destinations. Genome Biology 2004,5:109.
 Lloyd W. Sumner, David V. Huhman, Ewa Urbanczyk-Wochniak and Zhentian Lei: Methods, applications and
concepts of metabolite profiling: Secondary metabolism. Plant Biology Division 2007.
 Loo J.A., Udseth H.R. and Smith R.D. (June 1989). Peptide and protein analysis by electrospray ionization-
mass spectrometry and capillary electrophoresis-mass spectrometry. Anal Biochem 1989,179(2): 404–12.
 Marshall, A. G., Hendrickson, C. L. and Jackson, G. S: Fourier transform ion cyclotron resonance mass
spectrometry: a primer. Mass Spectrom. Rev.1998. 17 (1): 1–35.
 P. Krishnan, N. J. Kruger and R. G. Ratcliffe: Metabolite fingerprinting and profiling in plants using NMR.
Journal of Experimental Botany 2004, 56:410.
References

29. Reference contd.
 Rawi Ramautar Govert W. Somsen Gerhardus J. de Jong: CE-MS in metabolomics. Electropho
resis 2009,30:276-291.
 Ronald A. Hites: Gas Chromatography Mass Spectrometry. Indiana University School of Pub
lic and Environmental Affairs and Department of Chemistry, Chapter 31: 609-626.
 https://en.wikipedia.org/wiki/Capillary_electrophoresis%E2%80%93mass_spectrometry
 https://en.wikipedia.org/wiki/Nuclear_magnetic_resonance

30. THANK YOU VERY MUCH
FOR YOUR ATTENTION