Metabolimics

Question 1

What are metabolites?

Answer 1

Small molecule (metabolites) are biologic compounds which is lessthan 1000 m/z.

they are essential for their growth and maintenance of a biological system. Amino acids, carbohydrate, alcohols, lipids, vitamins (B2 and B12), polyols, organic acids, as well
as nucleotides (e.g. inosine-5’-monophosphate and guanosine-5’-monophosphate), Adenosine, mono, di Triphosphate.

Question 2

What is the difference between primary and secondary metabolism?

Answer 2

Primary metabolism:
Biomolecules which are synthesized by the cell because they are essential for their growth and maintenance of a biological system. Amino acids, carbohydrate, alcohols, lipids, vitamins

Secondary metabolites are organic compounds produced by bacteria, fungi, or plants which are not directly
involved in the normal growth, development, or reproduction of the organism. Unlike primary metabolites, absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism’ssurvivability

Question 3

Explain Warburg effect?

Answer 3

The Warburg effect refers to the observation that cancer cells preferentially produce energy through a process called anaerobic glycolysis, even when oxygen is present.

Through the study of metabolic changes in cancer cells, metabolomics can provide a deeper understanding of the Warburg effect, and potentially aid in the discovery of new therapies for cancer.

Question 4

Describe the subunits involved in genomics, Transcriptomics, proteomics and metabolimics?

Answer 4

Genomic (4 bases),
* Transcriptomics(4 bases),
* Protein (20 amino acids),
* Metabolomics(4-5 thousand metabolites across
multiple chemistry and chromatography)

Question 5

Define Metabolomics?

Answer 5

Metabolomics:The global characterization of all small molecules within a given biological system under specific
conditions.LC/MS High resolution, quantitation, (non volatile polar/non polar), GC/MS (volatiles), NMR

Question 6

Define Lipidomics?

Answer 6

Lipidomics: Analysis of all lipids, and the molecules with which they interact, and their function within biological
systems. (LC/MS) GC/MS.

Question 7

Define Metabolic profiling?

Answer 7

Identification and quantification of a selective number of predefined metabolites, which are generally
related to a specific metabolic pathway. (LC/MS, GC/MS, NMR)

Question 8

Define Metabolic fingerprinting

Answer 8

Global, high-throughput, rapid analysis to provide sample classification. Also utilized as a screening tool to discriminate between samples from different biological status or origin (i.e., case/control, disease/healthy). FTIR, NMR, GC/MS.
LC/MS, GC, LC.

Question 9

Define Metabolic foot printing

Answer 9

Analysis of the (exo) metabolites secreted/excreted by an organism; if the organism is
growing in culture this will include its environmental and growth substances. FTIR, NMR, GC/MS. LC/MS, GC, LC.

Question 10

Define Metabolite target analysis

Answer 10

Qualitative and quantitative analysis of one, or several, metabolites related to a specific
metabolic reaction (Hypothesis testing)

Question 11

Describe the steps in a metabolics experiment?

Answer 11

1. experiment design
2. sample preparation
3. sample analysis using MS and NMR
4. Data analysis
5. Metabolite identification

Question 12

describe Spectroscopic techniques (non destructive) within metabolomics?

Answer 12

Spectroscopic techniques are widely used in metabolomics to non-destructively analyze the metabolic profile of biological samples. These techniques are based on the absorption, emission, or scattering of electromagnetic radiation by molecules, and can provide information on the chemical composition and concentration of metabolites in a sample.

Nuclear magnetic resonance spectroscopy (NMR)
Fourier-transform infrared spectroscopy (FTIR)
Raman Spectroscopy (RS)
Surface-enhanced Raman scattering (SERS)
Surface-enhanced Resonant Raman scattering (SERRS)

Question 13

Explain Nuclear Magnetic Resonance (NMR) Spectroscopy?

Answer 13

Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR spectroscopy is a powerful technique that can be used to identify and quantify a wide range of metabolites in a sample. It is based on the absorption of radiofrequency radiation by the nuclei of certain atoms, and can provide information on the chemical structure, concentration, and environment of the metabolites.

Question 14

Decribe Raman Spectroscopy

Answer 14

Raman Spectroscopy: Raman spectroscopy is based on the scattering of light by molecules, and can provide information on the chemical structure and bonding of the sample. It is particularly useful for identifying and quantifying small molecules.

Question 15

Describe Fourier-transform infrared (FTIR) spectroscopy?

Answer 15

Fourier-transform infrared (FTIR) spectroscopy is a form of infrared (IR) spectroscopy that uses a Fourier transform to convert the infrared radiation absorbed by a sample into a spectrum that can be analyzed.

Question 16

Explain Quadrupole mass analyzer (QMF)

Answer 16

Quadrupole mass analyzer (QMF) is the simplest type of mass analyzer

Composed of 4 precisely arrange parallel electrodes. An electric field is generated within the space of the four electrodes using a specifically
applied voltage gradient to create an special type of electric field (quadrupolar field)
to scan the ions as it enter mass analyzer.

The voltage applied is 180o degree out of phase w.r.tto each and the voltage applied
is related to the ions of specific mass. (you can effectively dial ions of know mass in
and out of the QMF)

Therefore by the scanning the range of the voltage you can scan a specific massrange.

Question 17

How does electron volts effect fragmentation within a quadrapole?

Answer 17

In quadrupole mass spectrometry, fragmentation of molecules occurs when they are exposed to a high-energy environment.

Higher energy collisions (i.e. higher electron volts) generally result in more extensive fragmentation of the molecules. This is because the high-energy collision causes the bonds within the molecule to break, leading to the formation of smaller fragments.

Question 18

What is MS1 & 2

Answer 18

The ions that are produced in the MS1 stage are then selectively fragmented in a second stage of mass analysis, referred to as the “MS2” stage.

In the MS2 stage, the fragment ions are then analyzed by the mass spectrometer, and the resulting mass spectrum is called an MS2 spectrum.

Question 19

What usually follows mass spectrometry step in proteomics and metablomics?

Answer 19

Typically are connected to separation system (LC/GC) as part of an analytical
platform.

Question 20

Describe chromatography science?

Answer 20

Chromatography is the interaction of the complex mixture and its constituential components with the stationary phase SP (the column) and the mobile phase MP (gas in GC and solvents phase in LC respectively) to induced retention effectto separate the complex

Question 21

Explain how the plate theory relates to chromatography?

Answer 21

The plate theory is a theoretical model that describes the performance of chromatographic columns in terms of the number of theoretical plates. A theoretical plate is defined as a hypothetical zone in which a component of the mixture is separated from the others. The number of theoretical plates in a column is a measure of the efficiency of the separation and the higher the number of theoretical plates, the better the separation.

Question 22

Explain how Van Deemter equation relates to chromatography?

Answer 22

The Van Deemter equation is an empirical equation that describes the relationship between the efficiency of a chromatographic column and the flow rate of the mobile phase. The Van Deemter equation is based on the observation that the efficiency of a column is influenced by three main factors: eddy diffusion, longitudinal diffusion, and mass transfer. The Van Deemter equation expresses the relationship between these factors and the flow rate in a mathematical form.

The Van Deemter equation can be used to predict the effect of changes in the flow rate on the separation efficiency of a column and to optimize the flow rate for a given separation. By adjusting the flow rate, it is possible to achieve the best balance between the efficiency and the resolution of the separation

Question 23

Explain how Gas chromotography works?

Answer 23

The complex mixture isseparate in open tubular analytical column based on the gas phase chemistry and
temperature. (compounds within the complex mixture with the lowest boiling point will be eluted first)

As the gaseous sample is eluted from the column, the samples enter the ion source of the GC/MS.

Eluted samples are ionized using electron (EI), 70eV impact, induces mass fragmentation due to this high ev Low mass range and limited metabolite coverage 50-500 m/z.

Question 24

Describe the diffrence between Hilic and reverse phase chromatograhy

Answer 24

HILIC (Hydrophilic Interaction Chromatography) and reverse phase chromatography (RPC) are two different types of chromatography that are used to separate and purify small molecules.

HILIC is based on the interaction between the hydrophilic groups of the small molecules and a polar stationary phase, while RPC is based on the interaction between the hydrophobic groups of the small molecules and a non-polar stationary phase.

Question 25

Describe the difference between Hilic and reverse phase chromatography?

Answer 25

HILIC (Hydrophilic Interaction Chromatography) and reverse phase chromatography (RPC) are two different types of chromatography that are used to separate and purify small molecules.

HILIC is based on the interaction between the hydrophilic groups of the small molecules and a polar stationary phase, while RPC is based on the interaction between the hydrophobic groups of the small molecules and a non-polar stationary phase.

Question 26

Explain the principles of Liquid chromatography ?

Answer 26

Used to look at non-volatile compounds and based on high-pressure interactions within the stationary and liquid phase.

Different type of ionization (positive and negative mode) Eluted samples are ionized using electrospray ionization impact (2-4e KV),

A greater range of samples and sample matrix can be profiled using LC/based methods

Required DIA or DDA analysis for chemical identification

Very high metabolite coverage, higher mass range (100-1000 m/z)

Question 27

What factors should you consider when designing an experiment?

Answer 27

Is the equipment functioning optimally? - Run QC’s to test for variation.

Have you recorded enough data points to make this statistically valid? / definitively answer your hypothesis

Analysis tools? - : classification or calibration, cubic design, longitudinalstudy, multi class comparison?

Different omics has different constrains, which will dictate the number of total samples feasible

has the extraction methodology been optimised for the specific biological samples in
question?)

Question 28

Explain Unsupervised data analysis using PCA?

Answer 28

Unsupervised PCA is used when the goal is to simply identify the underlying structure of the data, without any reference to any known class labels or output values.

Question 29

Explain supervised data analysis using PCA?

Answer 29

Supervised PCA is used when the goal is to use the identified structure to predict class labels or output values. In this case, the algorithm tries to find the linear combinations of the original features that separate the different classes or that are most strongly correlated with the output values.