Metabolomics

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Metabolomics
Metabolomics is a novel “-omics” strategy, defined as the global or local profiling of metabolites in a given biological system, with broad coverage of different classes of endogenous metabolites. It is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind” or “Identification and quantification of the compounds (<1500 Da) in the metabolome”.
 
A metabolomics analysis provides a deeper understanding of metabolite expression within complex biological systems, and the proper analysis and interpretation of such results may potentially indicate the metabolite function.
 
At C-CAMP we have a well established Metabolomics Facility to help with identification and quantification of known compounds/metabolites in biological fluids such as Sera, Saliva, Tissue, Urine as well as from Cell extract using two parallel approaches like Targeted Metabolomics and Untargeted metabolite profiling (Comparative metabolomics) using High Resolution Mass Spectrometry (HRMS) system.
 


Resources for Metabolomics Platforms
Capabilities Expertise:
1. Full Scan (MS)/Product ion scan (MS/MS) analysis of known or purified compounds (Solid/Liquid)
 
This can be used to identify the precursor ions that yield a product ion for a specific m/z ratio
 
MS/MS analysis of Olanzapine. (A) UHPLC-MS chromatogram  of standard (olanzapine) and
internal standard  (olanzapine-d3) (B) MS/MS Spectrum of both standard (olanzapine ) and internal
standard (olanzapine-d3).
 
2. Analysis of compounds in biological matrix
 
(i)   Extraction of metabolites (liquid-liquid extraction, Solid phase extraction or both, depending on the nature of the metabolite)
(ii)  Analysis by Selected Reaction Monitoring (SRM) or Multiple Reaction Monitoring (MRM)
 
Absolute quantification of olanzapine and its metabolites using UHPLC-MS/SRM method. (A) Structures,
(B) UHPLC-MS/SRM chromatogram and (C) Standard curves for olanzapine and its metabolites.
 
3. Method development for specific metabolites to determine the absolute quantification
 
(i)   MS scan, MS/MS scan and picking the most intense ion for the MRM scan
(ii)  Spiking the standard and internal standard and extraction of metabolite from matrix (Sera, Saliva, Water, cell extract and Urine)
(iii) Detection of  Limit of Detection (LOD) and Limit of Quantification (LOQ)
(iv) Construction of standard curve
(v)  Validation of the method (Inter & Intraday variation, recovery, accuracy and precision)
(vi) Sample analysis
 
Absolute quantification of neurotransmitters from planarian extract using UHPLC-MS/SRM method.
(A) Picture of the intact planaria, (B) UHPLC-MS/SRM chromatogram of neurotransmitters standards and
internal standards and (C) comparison of neurotransmitters between sexual and asexual planaria.
 
4. Quantification of metabolites from plant extracts
 
Quantification of metabolite from plant extracts (dry powder from plant materials) using UHPLC-MS/SRM method
 
5. Metabolite Profiling/Comparative Metabolomics
Using Q-Exactive HRMS system we can now do the comparative metabolite profiling (Hydrophobic-reverse phase and Hydrophilic-HILIC) between control versus sample. The data analysis will be done using SIEVE 2.2 software using publically available data bases like HMDB and KEGG through Chemspider search.
 
Metabolite profiling workflow to analyze urine samples from nonsmokers and smokers. Samples were
prepared for two chromatographic separations and also for two ionization modes. Data analysis was carried out with SIEVE 2.2.
 
As part of the 'omics' technology platforms, C-CAMP via its collaborators also offers following services:
1. Data visualisation and reporting: After initial processing by the vendor specific software platforms, expertise can be availed for data cleaning, data transformations, statistical evaluations, visualisations, modelling and reporting 
2. Interactive visualisation and analysis via web application/dashboards. (For demo, visit https://www.sirpi.io/omicsvisdec)
3. Training on using the R programming packages : ready, tidyr, dplyr, ggplot2, plotly and the shiny package. 
Instruments

Methods Developed

Quantitative Metabolomics

S. No.

Compound Class

No.

Metabolites

1

Neurotransmitters

17

Histidine, Serine, Histamine, Aspartic acid, Glutamic acid, GABA, Nor-epinephrine, Dopa, Epinephrine, Octopamine, Tyrosine, Dopamine, Serotonin, Tyramine, Melatonin, Tryptophan, Tryptamine

2

Amino acid Panel

24

Histidine, Serine, Asparagine, Arginine, Aspartic acid, Glutamic acid, Threonine, Alanine, Proline, Tyrosine, Lysine, Methionine, Valine, Phenylalanine, Isoleucine, Tryptophan, Leucine, Taurine, Sarcosine, Citrulline, Ornithine, Homoserine, Aminoadipic acid, Hydroxyproline

3

Amines

37

Combination of Amino acids and Neurotransmitter Panels

4 Cholic acids Panel 14

GUDCA, GCA, TUDCA, TCA, CA, UDCA, GCDCA, GDCA, TDCA, GLCA, TLCA, CDCA, DCA, LCA

5

Nucleotides

4

 

6

Thiols

7

 

7

Plant Growth Hormones

13

Zeatin, Benzyl-aminopurine, Indole acetic acid, Indole propionic acid, Indole butyric acid, Gibberellic acid, Abscisic acid, Isopentenyl adenine, Salicylic acid, Jasmonic acid

8

Isoflavanoids and Phenolic acids

23

Quinic acid, Gallic acid, Chlorogenic acid, Catechin, Caffeic acid, Epicatechin, Epigallocatechin 3-gallate, umbelliferone, Liquiritin, Isovitexin, Isoquercetin, Genistin, Astragalin, Ononin, Luteolin, Quercetin, Naringenin, Kaempferol, Formononetin, Galangin, Licochalcone A, Glabridin

9

Plant Metabolites

9

Tulasi (Ocimum sanctum) and Neem (Azdirachta indica) Metabolites

10

Amyrins

2

 

11

Colistin from Sera

1

 

12

Olanzapine Metabolites

5

 

13

Folates

5

 

Metabolite Profiling

1

Comparative Metabolomics

Metabolite Profiling from Urine