Biomedical Analysis

2015/2016 First Semester

TAN Kui Thong
Office R724
Tel 35665
Email:kttan@mx.nthu.edu.tw
 Contents

• 1. Chromatography
• 2. Electrophoresis
• 3. Mass Spectrometry
• 4. Immunoassays
• 5. Quantitation of Total Proteins, Enzymes and Their Substrates
• 6. DNA isolation, Arrays and Sequencing
• 7. Protein Sequencing
• 8 Fluorescent Spectroscopy methods
• 9. Detection and Analysis by Fluorescent Reporters
• 10. Sensing Inside Living Cells and Tissues
 Textbooks

• Bioanalytical Chemistry, Andreas Manz, Nicole Pamme, Dimitri

Lossifidis, Imperial College Press.
• Basic Methods in Protein Purification and Analysis, Richard, J.
Simpson, Peter D. Adams, and Erica A. Golemis. CSH press.
• Clinical Chemistry, Principles, Techniques and Correlations, Michael
L. Bishop, Edward, P. Fody, Larry E. Schoeff, Walters Kluwer

Please visit http://lms.nthu.edu.tw/ occasionally for new handouts and

information.
 Examinations and Evaluation Method

• Midterm Exam (5th November 2015)

• Final Exam (7th January 2016)
• Midterm Exam: 50%
• Final Exam: 50%
• Bonus: 5% Attendance
Biomolecules
 Biomolecules

• A biomolecule is any molecule that is produced by a living organism.

• A diverse range of biomolecules exist, including:
• Small molecules:
– Lipids, polysaccharides, glycolipids, sterols, glycerolipids
– Vitamins
– Hormones, neurotransmitters
– Metabolites
– Natural products, secondary metabolites
• Monomers, oligomers and polymers:
 Carbohydrates
• The carbohydrates (saccharides) are divided into four chemical groupings:
monosaccharides, disaccharides, oligosaccharides, and polysaccharides. In general,
the monosaccharides and disaccharides, which are smaller carbohydrates, are
commonly referred to as sugars. The names of the monosaccharides and
disaccharides very often end in the suffix -ose. For example, grape sugar is the
monosaccharide glucose, cane sugar is the disaccharide sucrose.
• Polysaccharides serve for the storage of energy (e.g., starch and glycogen), and as
structural components (e.g., cellulose in plants and chitin in arthropods). The 5-
carbon monosaccharide ribose is an important component of coenzymes (e.g.,
ATP, FAD, and NAD) and the backbone of the genetic molecule known as RNA. The
related deoxyribose is a component of DNA. Saccharides and their derivatives
include many other important biomolecules that play key roles in the immune
system, fertilization, preventing pathogenesis, blood clotting, and development

Lactose is a disaccharide found in milk. It

consists of a molecule of D-galactose and a
molecule of D-glucose bonded by beta-1-4
glycosidic linkage.
 Lipids

• Lipids are a group of naturally occurring molecules that include fats, waxes, sterols,
fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides,
triglycerides, phospholipids, and others. The main biological functions of lipids
include storing energy, signaling, and acting as structural components of cell
membranes. Lipids have applications in the cosmetic and food industries as well as
in nanotechnology.

Cell membrane
 Amino Acids, Peptides and Proteins

• Amino acids are the building blocks for peptides and proteins and play
an important part in metabolism. 20 different amino acids are found in
living organisms. They can connect to each other via peptide bonds to
form long chains. Proteins may consist of thousands of amino acids
and can have molecular weights of up to several million Dalton (Da).
Shorter chains of up to a few hundred amino acids are referred to as
peptides. The sequence of the amino acids within the molecule is
essential for the structure and function of proteins and peptides in
biological processes.

Amino acid peptide Protein

 Amino Acids

• Amino acids can be classified according to their substituent R groups.

Basic Amino Acids Acidic amino acids

Secondary amino acid

Sulfur containing
amino acids
Aliphatic amino acids
Aromatic amino acids

Hydroxyl containing
amino acids
 Non-Standard Amino Acids

• Non-standard amino acids that are found in proteins are formed by post-
translational modification, which is modification after translation during protein
synthesis. These modifications are often essential for the function or regulation of
a protein; for example, the carboxylation of glutamate allows for better binding of
calcium cations, and the hydroxylation of proline is critical for maintaining
connective tissues.
• Some nonstandard amino acids are not found in proteins. Examples include
lanthionine, 2-aminoisobutyric acid, dehydroalanine, and the neurotransmitter
gamma-aminobutyric acid. Nonstandard amino acids often occur as intermediates
in the metabolic pathways for standard amino acids — for example, ornithine and
citrulline occur in the urea cycle, part of amino acid catabolism. A rare exception to
the dominance of α-amino acids in biology is the β-amino acid beta alanine (3-
aminopropanoic acid), which is used in plants and microorganisms in the synthesis
of pantothenic acid (vitamin B5), a component of coenzyme A.

GABA Carnitine α and β-alanine

 Zwitterionic character, pK and pI
• The carboxyl group of an amino acid has
a pK between 1.8 and 2.5, the amino
group has a pK between 8.7 and 10.7. At
the physiological pH 6 to 7, the amino
group is ionised to –NH3+ and the
carboxyl group is ionised to –COO−.

• For every amino acid, there is a specific

pH value at which it exhibits no net
charge. This is called the isoelectric point,
pI.
 Zwitterionic character, pK and pI

pI values of natural amino acids

pI values of some proteins

http://web.expasy.org/compute_pi/
 The Biological Function of Proteins

• Proteins have many different and varied biological functions and in addition to
their size, shape and orientation, can be classified according to their biological
roles within the cell.
 Structural Proteins: Ligaments contain elastin, which is a structural protein which can be
stretched in two dimensions. Hair, fingernails, feathers and horn all contain great
amounts of keratin, which is a tough, insoluble protein.
 Enzymes: Almost every chemical reaction between molecules in living cells are catalysed
by enzymes. Enzymes are the most varied and specialised proteins, and many thousands
of different types, each capable of catalysing a different type of chemical reaction. For
example, proteases to hydrolyse peptide bond.
 Nutrient and Storage Proteins: Ferritin, found in some bacteria, and also in plant and
animal tissues, stores iron. Ovalbumin was the major component protein of egg white
and casein was found in milk.
 Regulatory Proteins: Insulin, which regulates sugar metabolism and the growth
hormone secreted by the pituitary gland are two examples.
 Transport Proteins: In blood plasma, transport proteins bind and transport specific
molecules or ions from one organ to another. Haemoglobin in erythrocytes binds
oxygen as blood passes through the lungs, transporting it to the peripheral tissues, and
releases it to contribute in the energy-yielding oxidation of nutrients.
 The Primary Structure of Proteins
• The sequence of amino acids determines the primary structure of a protein.
Changing just a single amino acid in a critical position of the protein can
significantly alter its activity and function and be the cause of disease and
disorders. The amino acids are connected to each other by peptide bond.

(A) Peptide bond formation from two amino acids (B) Double bond character of the C-N
bond in a peptide

(C) The C-N bond is rigid due to the (D) A peptide with the sequence Ser-Ala-Cys-Gly
partial double bond character Showing N-Terminus and C-terminus.
 The Secondary Structure of Proteins

• The alpha helix is a common secondary structure of proteins and is a right-handed

coiled or spiral conformation (helix), in which every backbone N-H group donates a
hydrogen bond to the backbone C=O group of the amino acid.
• Beta sheets consist of beta strands connected laterally by at least two or three
backbone hydrogen bonds, forming a generally twisted, pleated sheet. A beta
strand is a stretch of polypeptide chain typically 3 to 10 amino acids long with
backbone in an almost fully extended conformation. The higher-level association
of β sheets has been implicated in formation of the protein aggregates and fibrils
observed in many human diseases, e.g. amyloidoses such as Alzheimer's disease.

(A) α-helix structure with the

–R group pointing outwards

(B) Hydrogen bonding

patterns of β-sheet
 The Tertiary Structure of Proteins

• The tertiary structure describes the complete three-dimensional structure of the

whole polypeptide chain. It includes the relationship of different domains formed
by the protein’s secondary structure and the interactions of the amino acid
substituent –R groups (H-bonding, hydrophobic interaction, disulfide bond). The
specific folding of a protein is only thermodynamically stable within a restricted
range of environmental parameters, i.e. the right temperature, pH and ionic
strength. Outside of this range, the protein could unfold and lose its activity.

(A) 3-D structure of ribonuclease H from E. Coli with α-helices and β-folding

(B) Formation of a disulfide bridge

 The quaternary Structure of Proteins

• A protein can consist of two or more separate polypeptide chains linked together.
Other, non-amino acid components such as minerals, lipids and carbohydrates can
also be part of a protein. The quaternary structure describes how these different
chains and components interact and connect to each other by hydrogen bonding,
electrostatic attraction and sulfide bridges.

(A) Insulin has one sulfide bridge within chain A

and two sulfide bridges between chain A and chain B

(B) Structure of haemoglobin

 Nucleic Acids
• Nucleic acids are polymeric macromolecules, or large biological molecules,
essential for all known forms of life. Nucleic acids, which include DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid), are made from nucleotide
monomers. Nucleic acids are found in abundance in all living things, where they
function in encoding, transmitting and expressing genetic information—in other
words, information is conveyed through the nucleic acid sequence, or the order of
nucleotides within a DNA or RNA molecule.

A comparison of the two principal nucleic

acids: RNA (left) and DNA (right), showing the
helices and nucleobases each employs.
 The Structure of Nucleic Acids

(A) Components of nucleic acids (D) The pentose β-D-ribose in RNA

and deoxy-ribose in DNA

(B) The structure of purine and derivatives

(E) Structure of nucleoside and nucleotide

(C) The structure of pyrimidine and derivatives

 3D Structure of DNA

(A) Structure and sequence direction of (B) DNA double helix

a DNA polynucleotide with the bases

(C) DNA base pairing.

Adenine and Thymine (A-T),
Guanine and Cytosine (G-C)
 Protein Synthesis

• Proteins are synthesized in the ribosomes of the cell cytoplasm. DNA, however, is
found in the cell nucleus. So how is the information contained in the DNA passed
out of the cell nucleus and into the cytoplasm.

(B) RNA is transcribed in the nucleus;

(A) Transcription of a DNA sequence into once completely processed, it is
mRNA followed by translation into a transported to the cytoplasm and
polypeptide sequence. translated by the ribosome (shown in
very pale grey behind the tRNA).
 The Genetic Code
• The genetic code is the set of rules by which information encoded within genetic
material (DNA or mRNA sequences) is translated into proteins by living cells. The
code defines how sequences of these nucleotide triplets, called codons, specify
which amino acid will be added next during protein synthesis. With some
exceptions, a three-nucleotide codon in a nucleic acid sequence specifies a single
amino acid.
 Parameters that concern Analytical Chemists

• Selectivity: For example, detection of a specific protein out of a proteins mixture.

• Sensitivity: The amount of analyte can be detected

• Time: How fast can the result obtained.

• Operation procedures: How complicated is the operating protocol.

• Sample: At what condition you want to measure the sample.

• Instrument: miniaturized equipment or big instrument.

Comparison of Classical and Bioanalytical Chemistry