Molecular Properties

Amino acids à Polypeptides à

primary, secondary, tertiary
structure à Protein.

ð Secondary structure:
Beta pleated sheets and alpha helix.

- 20 amino acids à 20n possible

sequences for polypeptide with
n amino acids.
- Wide variety of shapes and
structures depending on the
sequencing of amino acids.

Complexity of detecting
biomolecules:
- A human cell contains 10 bln
proteins.
Functional
identification of diversity
biomolecules

extracellular
signalling
human proteins activation
have 375 amino
acid residues

Numclear
Chromatography Mass
Magnetic
(LC/GC) Spectroscopy
Resonance

Liquide/Gas Chromatography
- Purification of large number of
compounds
- Common detectors:
ð Ultraviolet
ð Mass selective Detector
- Mobile phase: Gas such as helium.
- Stationary phase: High boiling point
liquid adsorbed onto a solid.
- Partition coefficient: The ratio of
conc. Of a neutral solute molecule
in a system with two immiscible
solvents.
Depends on:
ð Molecular size
ð Charge
ð Hydrophobicity
ð Specific binding interactions
- Tr – Retention time: the time b/w sample injection and an analyte peak reaching a detector at the end
of the column.
- Tm- Each analyte in a sample will have a different retention time. It is the time taken for the mobile
phase to pass through the column.
 Mass Spectrometry

Gas Phase Ionization

Electron Impact: For volatile
compounds and GCMS analysis Mass Analyzers: Tandem and Hybrid mass
ð Time-of-flight spectrometers:
Chemical Ionization: A softer
ionization for increasing molecular ð Quadrupole ð Triple quadrupole
ion production. ð Quadrupole ion Trap ð Quadrupole/time-of-
Ion (GCQ or LCQ) flight
Sources ð Linear quadrupole (LTQ) ð Tandem time-of-flight
Desorption Methods ð Fourier transform mass ð LTQ/Orbitrap
Matrix-assisted laser desorption spectrometer (FTMS)
(MALDI) ð Orbitrap
Electrospray Ionization: For the
analysis of proteins. peptides, carbs
and other biomacromolecules.

• Molecular weight is
determined from the
mass of a molecular
ion that appears in the
spectrum and is
directly related to the
 elemental composition of the compound.
• Fragment ions are produced whose masses are directly related to structure.
• For small molecules, we refer to this as an interpretive approach.
• For peptides, we refer to this a de novo sequencing.

Monoisotopic peak: Isotopically pure peak in the mass spectrum.

A higher proton affinity

means a more likely site for
protonation but may also
result in more
fragmentation.
Protonation of dipeptide à
cleavage of the amide bond.
 Nuclear Magnetic Resonance (NMR)
- The energy absorbed (or emitted) is in the form of electromagnetic (EM) radiation, like that in sunshine
and radio waves. According to Planck's Law, the energy of EM radiation is proportional to its frequency.
- NMR is based upon the concept that some atomic nuclei have an inherent angular momentum or
“spin” and there is an energy associated with each spin state.
- Proton - two spin states (+ 1/2 or -1/2)
- This gives rise to a magnetic moment for electrons and nuclei with spin angular momentum, since a
spinning charged particle must generate a magnetic field.

Larmor Frequency:
ð Under the influence of a
magnetic field, spins process about the
direction of the field at Larmor frequency,
which is proportional to the magnetic
field.
ð The distribution of nuclei b/w
the 2 spin states is determined by the
Boltzmann distribution.

Chemical shift:
ð All nuclei of a given nuclide would resonate at precisely the
same B0 for a given frequency.
Actually resonance occurs at
slightly different values of B0
for a given nucleus depending on its electron and molecular
environment.
ð These slight differences in absorption frequencies or chemical
shifts are what make NMR a valuable analytic tool.
 ð Chemical shifts arise because a nucleus is surrounded by an electron cloud. The charge distribution in
this cloud, and even electrons from neighboring atoms, can influence the magnetic field “felt” by the
nucleus, by generating small local magnetic fields

Factors affecting chemical shift:

1. Electronegative groups
2. Magnetic anisotropy of pi systems
3. Hydrogen bonding

HNMR spectrum of ethanol:

1. Chemical shifts are usually measured relative to the peak of position of an arbitrary reference
compound.
2. Tetramethyl silane (TMS) is conventionally used as a reference standard for proton and carbon
NMR.
3. Chemical shifts are measured as the distance between the observed peak position and TMS.
4. The source of peak splitting is a phenomenon called spin-spin coupling.

Technologies that will revolutionize healthcare:

- Artificial intelligence: Machine/software with the ability to depict or mimic human brain functions.
- 3D printing: tissue engineering
- Liquid Biopsy: Cancer detection
- Immunotherapy: cancer therapy
- CRISPR: gene editing

Omics:

- Omics aims at the collective characterization and quantification of pools of biological

molecules that translate into the structure, dynamics and function, of an organism.
- The first ‘omics’ term used was ‘genome’ created in 1920 by the botanist H. Winkler as
a blend of the words ‘gene’ and ‘chromosome’ to annotate the chromosome set as the
material foundations of an organism.
Human Genome Project:

1. In 1865, Gregor Mendel (Father of modern genetics) presents his research on experiments in plant
hybridization.
2. In 1869, Friedrich Miescher identifies “nuclein”, DNA with associated proteins, from cell nuclei.
3. Completed in April 2003

Genetics of cancer:

• Genes are found in the DNA in each cell that makes up your body. They control how the cell functions,
including how quickly it grows, how often it divides, and how long it lives. Researchers estimate that
there are 30,000 different genes in each cell.
• Genes are located on 46 chromosomes, which are arranged in two sets of 23 chromosomes.

CRISPR- Clustered Regularly Interspaced Short Palindromic Repeats (Gene Editing):

• Jennifer Doudna and Emmanuelle Charpentier co-discovered CRISPR, a gene-editing tool in 2012.
• CRISPR technology is a simple yet powerful tool for editing genomes. It allows researchers to easily
alter DNA sequences and modify gene function. Its many potential applications include correcting
genetic defects, treating and preventing the spread of diseases and improving crops.
• CRISPR can cut a string of DNA open. When that happens, DNA coding can be altered. And genes or
biological traits can be changed.
• These technologies allow genetic material to be added, removed, or altered at particular locations in
the genome.
• RISPR-Cas9, which is short for clustered regularly interspaced short palindromic repeats and CRISPR-
associated protein 9. The CRISPR-Cas9 system has generated a lot of excitement in the scientific
community because it is faster, cheaper, more accurate, and more efficient than other existing genome
editing methods.
• CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. The bacteria
capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR
arrays.
• CRISPR, the technique used by Chinese researcher He Jiankui to alter the DNA of Chinese twin girls Lula
and Nana, could introduce accidental mutations.
• Public opinion on gene editing to prevent disease is largely positive.
• When news broke last year that a Chinese biophysicist had used genome editing in an attempt to make
children more resistant to HIV, many scientists were quick to condemn the move as premature and
irresponsible.
• The most popular way to edit genes relies on a system called CRISPR–Cas9. Co-opted from a
mechanism that some microbes use to defend themselves against viruses, it uses an enzyme called
Cas9 to make cuts to DNA. A scientist can supply a snippet of RNA to guide Cas9 to a specific site in the
genome.
• But Cas9 and enzymes like it have been known to cut DNA at other sites, too, particularly when there
are DNA sequences in the genome similar to the target (see ‘Off-target effects’). Such ‘off target’ cuts
could result in health problems: a change to a gene that suppresses tumour growth, for example, might
lead to cancer.
• The single cell and single cell-type metabolomics approaches may allow researchers to overcome these
challenges in sampling through technologies such as iKnife or imaging mass spectrometry (MS)-based
efforts to capture metabolite signals.
• In addition to endogenous metabolites, other information from cells include oncometabolites,
exosome constituents, and epi-metabolites becomes available. This large scale unbiased metabolomics
datasets can help to paint a systems-scale picture with inputs from other -omics areas such as
genomics, transcriptomics and proteomics; they can also provide insightful biological data from cancers
and tumor tissues.
• Apply data analytics to make sense of this data and find robust and valid individual cancer- and tumor-
specific biomarkers and novel disease-specific pathways and networks. This ranges from machine to
deep learning methods popular in big data domains.

Genomics: deals with the entirety of an organism's hereditary information coded in its DNA (also called
genome).

Transcriptomics: deals with the entirety of RNA transcribed from the DNA (transcriptome).

Proteomics: deals with the entirety of proteins translated from the mRNA (proteome)

Epigenomics: addresses factors and mechanisms affecting the accessibility of genomic information by
modifications of its structure, e.g. via DNA-methylation or chemical modifications of the histones serving as
DNA-packing proteins (epigenome).

Microarray- High Throughput

A microarray is a multiplex lab-on-a-chip.

It is a 2D array on a solid substrate- usually
a glass slide of silicon thin-film cell- that
assays large amounts of biological
material using high-throughput screening
miniaturized, multiplexed and parallel
processing and detection methods.
Protein Microarray:

Protein microarrays are used to identify targets of drugs, small molecules, and enzyme substrates that may
be modified in numerous ways:

- By phosphorylation or methylation
- To detect protein binding properties
- To investigate protein-protein interactions
- To define protein- based biomarkers in a high throughput manner

Tissue microarray:

- To simultaneously probe 100s of human tissue cored by antibodies to detect protein abundance
(Immunohistochemistry), or by labelled nucleic acids (in situ hybridization) to detect transcript
abundance.
- Tissue microarray database (TMAD) archives multi-wavelength fluorescence and bright-field images of
tissue microarrays for scoring and analysis.
- We can examine the cells by staining in each tissue slice of the 3D Cultured tissue.

Cell Microarray:

- To study cell responses, immune cells.

- Arrays of peptide-major histocompatibility complexes (pMHC) together with antibodies against
secreted factors.
- T cell responses in patients undergoing a melanoma-associated peptide vaccine trial.
- Assessing pluripotency in human cells.

Antibody Microarray:

- Autoantibody profiling in multiple sclerosis using array of human protein fragments.

- Profiling the autoantibody repertoire with large antigen collections is emerging as a powerful tool for
the identification of biomarkers for autoimmune diseases.
- To screen for profiles of IgG in human plasma from individuals with multiple sclerosis related
diagnoses.
DNA microarrays:

- A tool used to determine whether the DNA from a particular individual contains a mutation in genes.
- To determine how often individuals with a particular mutation actually develop breast cancer, or to
identify the changes in gene sequences that are most often associated with particular diseases.
- Very large numbers of features can be put on microarray chips, representing a very large portion of the
human genome.

DNA

1. Two long strands, complimentary base pairing that makes

the shape of a double helix.
2. Anti-parallel strands of nucleotides.
3. Backbones of pentose sugars and phosphate groups.

Denature: double-helix DNA separates into 2 single-

strands) occurs at 95 ºC mimicking the function of helicase
in the cell.

Polymerase Chain Reaction

1. A small fragment of DNA section of interest needs to be

identified which serves as the template for producing the
primers that initiate the reaction.
2. 1 DNA molecule is used to amplify the DNA section.
3. DNA TEMPLATE: sample DNA containing the target
sequence. High temp. is applied to the original double stranded DNA molecule.
4. DNA POLYMERASE: to separate strands from each other and elongate the replication strand.
5. PRIMERS: short pieces of single-stranded DMA that are complimentary to the target sequence.
6. NUCLEOTIDES (dNTPs) PRIMER: Building blocked for new DNA.
7. RT PCR (reverse transcriptase PCR): PCR preceded with conversion of reverse transcriptase sample RNA
into cDNA with enzyme.
CT Contrast enhanced imaging:

PET Tracer:

- (18FTHK-5351) for tau pathology in Alzheimer’s disease

- 11C-Methionine -amino acid metabolism
- 8F-Fluorodeoxyglucose (FDG) -glucose metabolism

MRI:

- Linear MRI T1 contrast agent -DTPA

- Macrocyclic
MRI T1
contrast agent
-DOTA