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Protein sequencing, protein expression analysis using
protein microarray & protein localization using GFP
Submitted to,
Ms. Rishika P.S
Assistant Professor
Dept. of Botany
St. Teresa’s college
Ernakulam
Submitted by,
Silpa Selvaraj
Roll no: 13
II M.Sc. Botany
St. Teresa’s college
Ernakulam
GENOMICS,TRANSCRIPTOMICS, PROTEOMICS &
BIOINFORMATICS
OVERVIEW
2
• Protein sequencing.
• Protein expression analysis using protein microarray.
• Protein localization using GFP.
INTRODUCTION
3
• Protein sequencing refers to the methods for determining the amino acid sequence of proteins
and analysis of these sequences.
• Sequencing plays a very vital role in Proteomics as the information obtained can be used to
deduce function,structure, and location which in turn aids in identifying new or novel proteins as
well as understanding cellular processes.
• Better understanding of these processes allows for creation of drugs that target specific
metabolic pathways among other things.
• The general strategy for determining the amino acid sequence of a protein depends on whether:
the protein contains more than one polypeptide chain.
• If yes, then there is a need to separate and purify the chains.
• It is important to know disulfide bonds in the polypeptide chain must be cleaved.
4
• However, it is significant to check the composition of amino acids is determined in each polypeptide
chain.
• while performing protein sequencing it is essential to know and identify the amino acids at these
terminals.
• Proteins being macromolecules, we need to generate smaller fragments of peptides, so that the
analysis can be performed easily.
• The amino acid sequence (also called primary structure) of a protein is the order of the amino acids
in the protein linear chain.
HISTORY
5
• Sanger's first triumph was to determine the complete amino acid sequence of the two
polypeptide chains of bovine insulin, A and B, in 1951, .
• Prior to this it was widely assumed that proteins were somewhat amorphous.
• In determining these sequences, Sanger proved that proteins have a defined chemical
composition.
• In 1958, he was awarded a Nobel Prize in Chemistry for his work on the structure of proteins,
especially that of insulin.
• Sanger was given the challenge of determining amino acid sequence of insulin, which had never
been done before.
• Using chemistry and chromatography, and by mixing standard techniques with novel ones, he
developed a method to read the amino acid sequence of insulin and found that this protein is
actually made up of two polypeptide chains linked together by disulfide bonds.
STRUCTURE OF AMINO ACID
6
SANGER’S METHOD
7
• Sanger first needed to characterize the free amino groups in insulin.
• For this he developed a reagent, dinitrofluorobenzene (FDNB or DNFB, also called as
Sanger’s reagent).
• It reacted with amino groups present in proteins to form an acid-stable dinitrophrenyl (DNP)
derivative.
8
• The DNP protein was treated with acid to break the polypeptide backbone.
• The free DNP amino acid derivatives were isolated and compared to standards prepared from
known amino acids.
• In this way, Sanger determined that insulin was made up of two peptide chains: one (chain A)
with an aminoterminal glycine residue and another (chain B) with an amino-terminal
phenylalanine.
• Subsequent work revealed that chain A was composed of twenty amino acids and chain B thirty-
one.
• The individual chains were then broken down into smaller components:
• Acid was used to cleave the polypeptide backbone.
• Performic acid was used to break the cysteine disulfide bonds.
• Proteolytic enzymes were used to hydrolyze the polypeptide at specific sites on the chain.
• The reaction products were separated from each other and their sequence determined.
9
• Today, several refinement and
improvements have been made in
Sanger’s method.
• In this regard, sequencing of most
proteins can be performed within a few
hours or days using only a small amount
or microgram of protein.
EDMAN’S DEGRADATION METHOD
10
• Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in
a peptide.
• It is an N terminal sequence analysis method.
• In this method, the amino-terminal residue is labeled and cleaved from the peptide without
disrupting the peptide bonds between other amino acid residues.
• Automated edman sequencers are now in widespread use, and are able to sequence peptides up
to approximately 50 amino acids long.
MECHANISM
11
• Breakage of the disulfide bonds- by reducing agent= 2-mercaptoethanol.
• Protecting group – iodoacetic acid – prevents reformation of the bonds.
• Digestion into peptide fragments – trypsin/pepsin (endopeptidases), cyanogen bromide
(chemical agent).
• Adsorbtion of peptide to a solid surface – glass fibre coated with polybrene, porous
polyvinylidene fluoride (PVDF) membrane.
• Edman reagent, phenylisothiocyanate (PITC) is added to the adsorbed peptide along with a
mild basic buffer solution of 12% trimethylamine. This reacts with amine group of N terminal
amino acid to form a phenylthiocarbamoyl derivative.
• This phenylthiocarbamoyl derivative is then cleaved using Trifluoroacetic acid producing its
anilinothiazolinone derivative (ATZ-amino acid).
12
• The next terminal amino acid is now exposed and ready for the same reactions to occur.
• A wash is performed to remove excess buffers and reagents.
• ATZ residue is separated from the peptide by extraction in organic solvent (ethyl acetate or
chlorobutane), and is then converted to a more stable form phenylthiohydantoin (PTH).
• The PTH residue generated by each cycle is typically identified by chromatography, thin-layer
chromatography and reversed-phase high-performance liquid chromatography.
• The process can now be repeated for the remaining residues of the chain.
13
• Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been
chemically modified (e.g. by acetylation or formation of pyroglutamic acid).
• Sequencing will stop if a non-α-amino acid is encountered (e.g. isoaspartic acid).
• Edman degradation is generally not useful to determine the positions of disulfide bridges.
• It also requires peptide amounts of 1 picomole or above for discernible results.
• If the chain exceeds a length of 50-60 residues (30 residues in practice) the procedure tends to fail due to the
incompletion of the cyclical derivitization.
• This can be solved by taking the larger peptide chain and cleaving it into smaller fragments using cyanogen bromide,
trypsin, chemotrypsin or any enzyme/chemical which can break peptide chains.
• An advantage of the Edman degradation is that it only uses 10 - 100 pico-moles of peptide for the sequencing process.
The Edman degradation reaction was automated in 1967 by Edman and Beggs to speed up the process and 100
automated devices were in use worldwide by 1973.
MASS SPECTROMETRY
14
Mass Spectrometry (MS) is an analytical chemistry technique that helps identify the amount
and type of chemicals present in a sample by measuring the mass-to-charge ratio.
In this instrumental technique, the sample is converted to rapidly moving positive ions by
electron bombardment and charged particles are separated according to their masses.
15
• In this technique, molecules are bombarded with a beam
of energetic electrons.
• The molecules are ionized and broken up into many
fragments, some of which are positive ions.
• Each ind of ion has a particular ratio of mass to charge,
i.e. m/e ratio (value).
• The ions pass through magnetic and electric fields to
reach the detector where they are detected and signals
are recorded to give mass spectra.
PRINCIPLE
WORKING
16
• A sample, which may be solid, liquid, or gas, is ionized, for example by bombarding it with
electrons.
• This may cause some of the sample’s molecules to break into positively charged fragments.
• These ions are then separated according to their mass-to-charge ratio, typically by accelerating
them and subjecting them to an electric or magnetic field: Ions of the same mass-to-charge
ratio will undergo the same amount of deflection.
• The ions are detected by a mechanism capable of detecting charged particles, such as
an electron multiplier.
• Results are displayed as spectra of the relative abundance of detected ions as a function of the
mass-to-charge ratio.
• The atoms or molecules in the sample can be identified by correlating known masses (e.g. an
entire molecule) to the identified masses or through a characteristic fragmentation pattern.
17
Sample Inlet
A sample stored in the large reservoir from which molecules reach the ionization chamber at low
pressure in a steady stream by a pinhole called “Molecular leak”.
Ionization
Atoms are ionized by knocking one or more electrons off to give positive ions by bombardment with
a stream of electrons. Most of the positive ions formed will carry a charge of +1.
Ionization can be achieved by :
Electron Ionization (EI-MS)
Chemical Ionization (CI-MS)
Desorption Technique (FAB)
18
Acceleration
• Ions are accelerated so that they all have the same kinetic energy.
• Positive ions pass through 3 slits with voltage in decreasing order.
• Middle slit carries intermediate and finals at zero volts.
Deflection
• Ions are deflected by a magnetic field due to differences in their masses.
• The lighter the mass, the more they are deflected.
• It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, the more it
will be deflected.
19
Detection
• The beam of ions passing through the mass analyzer is detected by a detector on the basis of
the m/e ratio.
• When an ion hits the metal box, the charge is neutralized by an electron jumping from the
metal onto the ion.
• Types of analyzers:
• Magnetic sector mass analyzers
• Double focussing analyzers
• Quadrupole mass analysers
• Time of Flight analyzers (TOF)
• Ion trap analyzer
• Ion cyclotron analyser
20
Applications of Mass Spectrometry (MS)
• Environmental monitoring and analysis (soil, water, and air pollutants, water quality, etc.)
• Geochemistry – age determination, soil, and rock composition, oil and gas surveying
• Chemical and Petrochemical industry – Quality control
• Identify structures of biomolecules, such as carbohydrates, nucleic acids
• Sequence biopolymers such as proteins and oligosaccharides
• Determination of the molecular mass of peptides, proteins, and oligonucleotides.
• Monitoring gases in patients’ breath during surgery.
• Identification of drug abuse and metabolites of drugs of abuse in blood, urine, and saliva.
• Analyses of aerosol particles.
• Determination of pesticides residues in food.
•
a)Mass spectrometry is quickly becoming the gold standard by which to
identify protein sequences due to its ease of automation and extreme
accuracy.
c)The two most popular methods to identify protein sequences using Mass
Spectrometry are Peptide Mass Fingerprinting and Tandem Mass
Spectrometry.
21
PEPTIDE MASS FINGERPRINTING
22
• Peptide mass fingerprinting (PMF), also known as protein fingerprinting, is an analytical
technique for protein identification in which the unknown protein of interest is first cleaved into
smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such
as MALDI-TOF or ESI-TOF.
• The method was developed in 1993 by several groups independently.
• The peptide masses are compared to either a database containing known protein sequences or even
the genome.
• This is achieved by using computer programs that translate the known genome of the organism into
proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the
peptides from each protein.
• The results are statistically analyzed to find the best match.
• The advantage of this method is that only the masses of the peptides have to be known. A
disadvantage is that the protein sequence has to be present in the database of interest.
23
• The peptides are then dissolved in a small amount of distilled water or further concentrated and purified
and are ready for mass spectrometric analysis.
• The digested protein can be analyzed with different types of mass spectrometers such as ESI-TOF
or MALDI-TOF.
• MALDI-TOF is often the preferred instrument because it allows a high sample throughput and several
proteins can be analyzed in a single experiment.
• The proteins are cut into several fragments using proteolytic enzymes such
as trypsin, chymotrypsin or Glu-C. A typical sample:protease ratio is 50:1.
• The proteolysis is typically carried out overnight and the resulting peptides are extracted
with acetonitrile and dried under vacuum.
• The mass spectrometric analysis produces a list of molecular weights which is often called a peak list.
The peptide masses are compared to protein databases such as Swissprot, which contain protein
sequence information. Software performs in silico digests on proteins in the database with the same
enzyme (e.g. trypsin) used in the chemical cleavage reaction. The mass of these peptides is then
calculated and compared to the peak list of measured masses.
24
• A small fraction of the peptide (usually 1 microliter or less) is pipetted onto a MALDI target and a
chemical called a matrix is added to the peptide mix.
• Common matrices are sinapinic acid, Alpha-Cyano-4-hydroxycinnamic acid, and 2,3-
Dihydroxybenzoic acid. The matrix molecules are required for the desorption of the peptide
molecules.
• Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed.
• MALDI-MS sample preparation technique; dried droplet technique; The target is inserted into the
vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide
fragments is initiated by a pulsed laser beam which transfers high amounts of energy into the matrix
molecules.
• The energy transfer is sufficient to promote the ionisation and transition of matrix molecules and
peptides from the solid phase into the gas phase.
• The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector
where their arrival is detected as an electric signal.
TANDEM MASS SPECTROMETRY
25
ANALYSIS OF C TERMINAL AMINOACIDS
26
• Fewer methods of analysis are available for the identification of C-terminal residue of polypeptides.
Hydrazinolysis:
• Proteins are treated with anhydrous hydrazine (H2N-NH2) at 100° C.
• So, all the amino acids of polypeptide chain is reacted with hydrazine and converted into amino
acid hydrazides.
• But, the amino acid attached to carboxyl terminal is not reacted and remain as the free amino acid.
• This free amino acid can be isolated and identified by chromatographic analysis.
27
LiAlH4 (Lithium aluminium hydride) based reduction of C-terminus:
• Proteins are analysed by this method through the process of reduction.
• Reduction of free α-amino group at the end of a peptide chain into an alcohol is performed by LiAlH4.
• After the reduction process, subsequent acid hydrolysis of protein is carried out and free-amino acids
with a single free alcohol are obtained.
• This single free alcohol represented the C-terminal residue.
Enzymatic analysis or carboxypeptidase-based analysis:
• Most recent and popular method of analysis of C-terminus of protein is the enzymatic analysis or
carboxypeptidase-based analysis.
• The enzyme carboxypeptidase cut the amino acid residues from the C-terminus of a protein in a
sequential way.
• Free amino acids from C-terminus of a protein can be isolated and identified by chromatographic
analysis.
SIGNIFICANCE OF PROTEIN SEQUENCING
28
• Information of the sequence of a protein is very essential in explaining its mechanism of
action.
• The three-dimensional structure of proteins is explained by the information of amino acid
sequence.
• Amino acid sequence provides a relation between the genetic message in RNA and three-
dimensional structure that performs a proteins biological function.
• Amino acid sequence determination is an essential part of molecular pathology.
• Alterations in amino acid sequence can produce abnormal function of protein and resulting in
disease development, such as
• sickle cell anemia & cystic fibrosis.
• Evolutionary history is also determined by protein sequencing.
• Protein resembles with another amino acid sequence only if they have a common ancestor.
Therefore, molecular events in evolution can be traced from amino acid sequences.
PROTEIN MICROARRAY
29
• A protein microarray (or protein chip) is a high-throughput method used to track the
interactions and activities of proteins, and to determine their function, and determining
function on a large scale.
• Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel.
The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead,
or microtitre plate, to which an array of capture proteins is bound.
• Probe molecules, typically labeled with a fluorescent dye, are added to the array.
• Any reaction between the probe and the immobilised protein emits a fluorescent signal that is
read by a laser scanner.
• Protein microarrays are rapid, automated, economical, and highly sensitive.
• The proteins are arrayed onto a solid surface
such as microscope slides, membranes, beads
or microtitre plates.
• The function of this surface is to provide a
support onto which proteins can be
immobilized, maximal binding properties,
whilst maintaining the protein in its native
conformation so that its binding ability is
retained.
• Microscope slides made of glass or silicon are
a popular choice since they are compatible
with the easily obtained robotic arrayers and
laser scanners that have been developed for
DNA microarray technology.
• Nitrocellulose film slides are broadly accepted
as the highest protein binding substrate for
protein microarray applications.
METHOD
• The chosen solid surface is then covered with a
coating that must serve the simultaneous
functions of immobilising the protein,
preventing its denaturation, orienting it in the
appropriate direction so that its binding sites are
accessible, and providing
a hydrophilic environment in which the binding
reaction can occur.
• Immobilising agents include layers of
aluminium or gold, hydrophilic polymers,
and polyacrylamide gels, or treatment
with amines, aldehyde or epoxy.
• Thin-film technologies like physical vapour
deposition (PVD) and chemical vapour
deposition (CVD) are employed to apply the
coating to the support surface.
30
• In the most common type of protein array, robots
place large numbers of proteins or
their ligands onto a coated solid support in a pre-
defined pattern. This is known as robotic contact
printing or robotic spotting.
• Another method is ink-jetting, non-contact method
of dispersing the protein polymers onto the solid
surface in the desired pattern.
• Piezoelectric spotting is a similar to ink-jet
printing. The printhead moves across the array, and
at each spot uses electric stimulation to deliver the
protein molecules onto the surface via tiny jets.
This is also a non-contact process.
• Photolithography is a fourth method of arraying the
proteins onto the surface. Light is used in
association with photomasks.
• The most widely used method for detection is
fluorescence labeling which is highly
sensitive, safe and compatible with readily
available microarray laser scanners.
• Other labels can be used, such as affinity,
photochemical or radioisotope tags.
• These labels are attached to the probe itself
and can interfere with the probe-target protein
reaction.
• Label free detection methods; surface plasmon
resonance (SPR), carbon nanotubes, carbon
nanowire sensors (where detection occurs via
changes in conductance) and
microelectromechanical system (MEMS)
cantilevers.[
31
An aqueous environment is essential at all stages to prevent protein denaturation. Therefore,
sample buffers contain a high percent of glycerol (to lower the freezing point), and the
humidity of the manufacturing environment is carefully regulated. Microwells have the dual
advantage of providing an aqueous environment while preventing cross-contamination
between samples.
PROCEDURE
32
Selection of Proteins:
• Choose a set of proteins relevant to the research question or experiment. These can be purified proteins,
protein fragments, or even whole-cell lysates.
Microarray Substrate Preparation:
• Select a solid support for the microarray, such as a glass slide, membrane, or a specialized chip.
• Clean the substrate to ensure a proper surface for protein immobilization.
Immobilization of Proteins:
• Choose an immobilization method based on the microarray substrate and experimental requirements.
• Spotting: Use robotic spotters or printers to deposit small droplets of protein solutions onto the substrate.
• In Situ Synthesis: Utilize techniques like photochemical reactions or inkjet printing to synthesize proteins
directly on the substrate.
• Surface Immobilization: Covalently attach proteins to the substrate through chemical reactions or
physically adsorb them.
33
Blocking and Stabilization:
• Block the remaining active sites on the substrate to prevent non-specific binding of proteins
during subsequent steps.
• Stabilize the immobilized proteins to maintain their activity and conformation.
Sample Incubation:
• Incubate the microarray with a biological sample containing proteins of interest (e.g., cell
lysates, purified proteins, or serum).
• Allow time for specific interactions to occur between the immobilized proteins and those in the
sample.
Washing:
• Wash the microarray to remove unbound or nonspecifically bound proteins, reducing
background noise.
• Use buffer solutions with appropriate detergents and salts for efficient washing.
34
Detection Method:
• Choose a suitable detection method based on the experimental goals and the nature of the proteins
being studied.
Fluorescence Detection:
• Label proteins with fluorescent dyes or tags.
• Use a fluorescence scanner to visualize and quantify signals.
Chemiluminescence:
• Utilize enzymatic reactions to produce light.
• Image chemiluminescent signals using specialized equipment.
Radioactive Labeling:
• Label proteins with radioactive isotopes (less common due to safety concerns).
• Use autoradiography for detection.
35
Data Acquisition:
• Capture images or signals generated by the detection method.
• Collect data on the intensity, location, and patterns of signals from the microarray.
Data Analysis:
• Analyze the data using bioinformatics tools and software.
• Interpret the results, identify protein interactions, assess expression levels, or investigate post-
translational modifications.
Validation:
• Validate the results using additional experimental techniques, such as immunoprecipitation,
Western blotting, or other assays, to confirm the observed protein interactions.
Interpretation and Conclusion:
• Draw conclusions based on the analyzed data.
• Discuss the biological significance of the findings and their implications for the research question.
APPLICATIONS OF PROTEIN MICROARRAY
36
Disease Biomarker Discovery:
• Objective: Identify protein biomarkers associated with diseases.
• Application: Early detection, prognosis, and monitoring of diseases such as cancer,
neurodegenerative disorders, and autoimmune diseases.
Diagnostic Tools in Medicine:
• Objective: Develop diagnostic assays for diseases.
• Application: Protein microarrays can be used for clinical diagnostics, allowing for rapid and
multiplexed detection of disease markers in patient samples.
Drug Target Discovery and Validation:
• Objective: Identify potential drug targets and validate their relevance.
• Application: Accelerate drug discovery by profiling proteins to discover targets, assess drug
interactions, and validate potential therapeutic candidates.
37
Pharmacogenomics:
• Objective: Personalize drug treatments based on individual genetic variations.
• Application: Identify patient-specific protein expression patterns to predict responses to
drugs, optimize dosages, and minimize adverse effects.
Protein-Protein Interaction Studies:
• Objective: Investigate interactions between proteins.
• Application: Understand cellular signaling pathways, protein complexes, and regulatory
networks, providing insights into cellular functions.
Functional Proteomics:
• Objective: Study the function of proteins in a systematic manner.
• Application: Characterize the roles of proteins in cellular processes, signaling cascades, and
pathways to gain a comprehensive understanding of cellular function.
38
Autoimmune Disease Research:
• Objective: Investigate autoantibody profiles and protein targets in autoimmune diseases.
• Application: Uncover autoimmune disease mechanisms, identify autoantigens, and develop
diagnostic tools for autoimmune conditions.
Allergy Profiling:
• Objective: Identify allergenic proteins and study allergic responses.
• Application: Characterize allergic reactions by detecting specific IgE antibodies against
allergens, aiding in allergy diagnosis and treatment.
Vaccine Development:
• Objective: Identify immunogenic proteins for vaccine candidates.
• Application: Screen and identify potential vaccine antigens by assessing antibody responses
and epitope mapping.
39
Pathogen Detection and Characterization:
• Objective: Identify and characterize proteins from pathogens.
• Application: Develop diagnostic tools for infectious diseases, study pathogen-host interactions,
and identify potential vaccine targets.
Stem Cell Research:
• Objective: Study protein expression profiles during stem cell differentiation.
• Application: Understand the molecular mechanisms involved in stem cell development and
differentiation.
Environmental Monitoring:
• Objective: Detect and quantify proteins indicative of environmental conditions or pollution.
• Application: Monitor environmental changes, assess pollution levels, and study the impact on
living organisms.
40
Environmental Monitoring:
• Objective: Detect and quantify proteins indicative of environmental conditions or pollution.
• Application: Monitor environmental changes, assess pollution levels, and study the impact on
living organisms.
Neuroscience Research:
• Objective: Investigate protein expression patterns in the nervous system.
• Application: Study neurological disorders, neuronal development, and synaptic functions.
Cell Signaling Pathway Analysis:
• Objective: Explore signaling pathways and protein cascades.
• Application: Investigate how proteins communicate within cells, helping to understand
cellular responses to stimuli and external factors.
ANALYTICAL PROTEIN MICROARRAY
41
• Analytical microarray are also known as capture arrays, uses a library
of antibodies, aptamers or affibodies arrayed on the support surface.
• These are used as capture molecules since each binds specifically to a
particular protein.
• The array is probed with a complex protein solution such as a cell
lysate.
• This type of microarray is especially useful in comparing protein
expression in different solutions.
• For instance the response of the cells to a particular factor can be
identified by comparing the lysates of cells treated with specific
substances or grons under certain conditions with the lysates of control
cells.
• Another application is in the identification and profiling of diseased
tissues.
Types of protein microarray
FUNCTIONAL PROTEIN MICROARRAY
42
• Functional protein microarrays (also known as target protein arrays)
are constructed by immobilizing large numbers of purified proteins
and are used to identify protein-protein, protein-DNA, protein-RNA,
protein-phospholipid and protein-small- molecule interactions, to
assay enzymatic activity and to detect antibodies and demonstrate
their specificity.
• They differ from analytical arrays in that functional protein arrays are
composed of arrays containing full-length functional proteins or
protein domains.
• These protein chips are used to study the biochemical activities of the
entire proteome in a single experiment.
GFP TAGGING
43
• Green Fluorescent Protein (GFP) was first isolated from the jelly fish, Aequorea victoria.
• The protein has 238 amino acids, three of them (65 to 67) form a structure that emits visible green
fluorescent light when exposed to UV light.
• Biologists use GFP to study cells in embryos and fetuses during developmental processes and also as a
marker protein.
• GFP can attach to and mark another protein with fluorescence, enabling scientists to see the presence of
the particular protein in an organic structure.
• Using rDNA technology, scientists combine the gfp gene to another gene that produces a protein that
they want to study, and then they insert the complex into a cell.
• If the cell produces the green fluorescence, scientists infer that the cell expresses the target gene.
• Moreover, scientists use GFP to label specific organelles, cells, tissues.
44
• As the Gfp gene is heritable, the descendants of labeled entities also exhibit green
fluorescence.
• The gene for GFP was successfully inserted into E.coli bacteria in 1994 and the Nobel Prize in
Chemistry 2008 was jointly awarded to Osamu Shimomura, Martin Chalfie and Roger Y. Tsien
for the discovery and development of GFPs.
• GFP is generally non-toxic and can be expressed to high levels in different organisms with
minor effects on their physiology.
• When the gene for GFP is fused to the gene of a protein to be studied, the expressed protein of
interest retains its normal activity.
• Likewise, GFP retains its fluorescence, so that the location, movement and other activities of
the studied protein can be followed by microscopic monitoring of the GFP fluorescence
STRUCTURE OF GFP
45
• The structure includes primary structure of a chain of 238 amino acids,
a secondary structure of hydrogen-bonded helices and pleated sheets
and a tertiary barrel shaped structure capped with helices.
• At the center - chromophore, an amino acid section responsible for the
light emission.
• Particularly useful for fluorescence microscopy as the chromophore is
encased in the barrel shaped structure, avoiding solvents and allowing
the protein to fluoresce in many conditions.
• Unlike other proteins that require enzymes for the complex folding
necessary for the required protein shape, this occurs automatically in
GFP.
REFERENCES
46
• eGyanKosh https://egyankosh.ac.in › unit-6PDF SEQUENCING AND ANALYSIS OF
PROTEIN.
• Future Science https://www.future-science.com › doi Protein Microarrays | BioTechniques
• Mark Schena (2005). Protein Microarrays Jones & Bartlett Learning. pp. 47–. ISBN 978-0-
7637-3127-4
• Remington S. J. (2011). Green fluorescent protein: a perspective. Protein science : a
publication of the Protein Society, 20(9), 1509–1519. https://doi.org/10.1002/pro.684
• UPV/EHU https://www.ehu.eus › papersPDF Peptide Sequencing by Edman Degradation.
• Zou, Yawen, "Green Fluorescent Protein”. Embryo Project Encyclopedia ( 2014-06-11 ). ISSN:
1940-5030 https://hdl.handle.net/10776/7903
THANK YOU
47

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Protein sequencing. Protein expression analysis using protein microarray. Protein localization using GFP.

  • 1. Protein sequencing, protein expression analysis using protein microarray & protein localization using GFP Submitted to, Ms. Rishika P.S Assistant Professor Dept. of Botany St. Teresa’s college Ernakulam Submitted by, Silpa Selvaraj Roll no: 13 II M.Sc. Botany St. Teresa’s college Ernakulam GENOMICS,TRANSCRIPTOMICS, PROTEOMICS & BIOINFORMATICS
  • 2. OVERVIEW 2 • Protein sequencing. • Protein expression analysis using protein microarray. • Protein localization using GFP.
  • 3. INTRODUCTION 3 • Protein sequencing refers to the methods for determining the amino acid sequence of proteins and analysis of these sequences. • Sequencing plays a very vital role in Proteomics as the information obtained can be used to deduce function,structure, and location which in turn aids in identifying new or novel proteins as well as understanding cellular processes. • Better understanding of these processes allows for creation of drugs that target specific metabolic pathways among other things. • The general strategy for determining the amino acid sequence of a protein depends on whether: the protein contains more than one polypeptide chain. • If yes, then there is a need to separate and purify the chains. • It is important to know disulfide bonds in the polypeptide chain must be cleaved.
  • 4. 4 • However, it is significant to check the composition of amino acids is determined in each polypeptide chain. • while performing protein sequencing it is essential to know and identify the amino acids at these terminals. • Proteins being macromolecules, we need to generate smaller fragments of peptides, so that the analysis can be performed easily. • The amino acid sequence (also called primary structure) of a protein is the order of the amino acids in the protein linear chain.
  • 5. HISTORY 5 • Sanger's first triumph was to determine the complete amino acid sequence of the two polypeptide chains of bovine insulin, A and B, in 1951, . • Prior to this it was widely assumed that proteins were somewhat amorphous. • In determining these sequences, Sanger proved that proteins have a defined chemical composition. • In 1958, he was awarded a Nobel Prize in Chemistry for his work on the structure of proteins, especially that of insulin. • Sanger was given the challenge of determining amino acid sequence of insulin, which had never been done before. • Using chemistry and chromatography, and by mixing standard techniques with novel ones, he developed a method to read the amino acid sequence of insulin and found that this protein is actually made up of two polypeptide chains linked together by disulfide bonds.
  • 7. SANGER’S METHOD 7 • Sanger first needed to characterize the free amino groups in insulin. • For this he developed a reagent, dinitrofluorobenzene (FDNB or DNFB, also called as Sanger’s reagent). • It reacted with amino groups present in proteins to form an acid-stable dinitrophrenyl (DNP) derivative.
  • 8. 8 • The DNP protein was treated with acid to break the polypeptide backbone. • The free DNP amino acid derivatives were isolated and compared to standards prepared from known amino acids. • In this way, Sanger determined that insulin was made up of two peptide chains: one (chain A) with an aminoterminal glycine residue and another (chain B) with an amino-terminal phenylalanine. • Subsequent work revealed that chain A was composed of twenty amino acids and chain B thirty- one. • The individual chains were then broken down into smaller components: • Acid was used to cleave the polypeptide backbone. • Performic acid was used to break the cysteine disulfide bonds. • Proteolytic enzymes were used to hydrolyze the polypeptide at specific sites on the chain. • The reaction products were separated from each other and their sequence determined.
  • 9. 9 • Today, several refinement and improvements have been made in Sanger’s method. • In this regard, sequencing of most proteins can be performed within a few hours or days using only a small amount or microgram of protein.
  • 10. EDMAN’S DEGRADATION METHOD 10 • Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. • It is an N terminal sequence analysis method. • In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues. • Automated edman sequencers are now in widespread use, and are able to sequence peptides up to approximately 50 amino acids long.
  • 11. MECHANISM 11 • Breakage of the disulfide bonds- by reducing agent= 2-mercaptoethanol. • Protecting group – iodoacetic acid – prevents reformation of the bonds. • Digestion into peptide fragments – trypsin/pepsin (endopeptidases), cyanogen bromide (chemical agent). • Adsorbtion of peptide to a solid surface – glass fibre coated with polybrene, porous polyvinylidene fluoride (PVDF) membrane. • Edman reagent, phenylisothiocyanate (PITC) is added to the adsorbed peptide along with a mild basic buffer solution of 12% trimethylamine. This reacts with amine group of N terminal amino acid to form a phenylthiocarbamoyl derivative. • This phenylthiocarbamoyl derivative is then cleaved using Trifluoroacetic acid producing its anilinothiazolinone derivative (ATZ-amino acid).
  • 12. 12 • The next terminal amino acid is now exposed and ready for the same reactions to occur. • A wash is performed to remove excess buffers and reagents. • ATZ residue is separated from the peptide by extraction in organic solvent (ethyl acetate or chlorobutane), and is then converted to a more stable form phenylthiohydantoin (PTH). • The PTH residue generated by each cycle is typically identified by chromatography, thin-layer chromatography and reversed-phase high-performance liquid chromatography. • The process can now be repeated for the remaining residues of the chain.
  • 13. 13 • Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminus has been chemically modified (e.g. by acetylation or formation of pyroglutamic acid). • Sequencing will stop if a non-α-amino acid is encountered (e.g. isoaspartic acid). • Edman degradation is generally not useful to determine the positions of disulfide bridges. • It also requires peptide amounts of 1 picomole or above for discernible results. • If the chain exceeds a length of 50-60 residues (30 residues in practice) the procedure tends to fail due to the incompletion of the cyclical derivitization. • This can be solved by taking the larger peptide chain and cleaving it into smaller fragments using cyanogen bromide, trypsin, chemotrypsin or any enzyme/chemical which can break peptide chains. • An advantage of the Edman degradation is that it only uses 10 - 100 pico-moles of peptide for the sequencing process. The Edman degradation reaction was automated in 1967 by Edman and Beggs to speed up the process and 100 automated devices were in use worldwide by 1973.
  • 14. MASS SPECTROMETRY 14 Mass Spectrometry (MS) is an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio. In this instrumental technique, the sample is converted to rapidly moving positive ions by electron bombardment and charged particles are separated according to their masses.
  • 15. 15 • In this technique, molecules are bombarded with a beam of energetic electrons. • The molecules are ionized and broken up into many fragments, some of which are positive ions. • Each ind of ion has a particular ratio of mass to charge, i.e. m/e ratio (value). • The ions pass through magnetic and electric fields to reach the detector where they are detected and signals are recorded to give mass spectra. PRINCIPLE
  • 16. WORKING 16 • A sample, which may be solid, liquid, or gas, is ionized, for example by bombarding it with electrons. • This may cause some of the sample’s molecules to break into positively charged fragments. • These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: Ions of the same mass-to-charge ratio will undergo the same amount of deflection. • The ions are detected by a mechanism capable of detecting charged particles, such as an electron multiplier. • Results are displayed as spectra of the relative abundance of detected ions as a function of the mass-to-charge ratio. • The atoms or molecules in the sample can be identified by correlating known masses (e.g. an entire molecule) to the identified masses or through a characteristic fragmentation pattern.
  • 17. 17 Sample Inlet A sample stored in the large reservoir from which molecules reach the ionization chamber at low pressure in a steady stream by a pinhole called “Molecular leak”. Ionization Atoms are ionized by knocking one or more electrons off to give positive ions by bombardment with a stream of electrons. Most of the positive ions formed will carry a charge of +1. Ionization can be achieved by : Electron Ionization (EI-MS) Chemical Ionization (CI-MS) Desorption Technique (FAB)
  • 18. 18 Acceleration • Ions are accelerated so that they all have the same kinetic energy. • Positive ions pass through 3 slits with voltage in decreasing order. • Middle slit carries intermediate and finals at zero volts. Deflection • Ions are deflected by a magnetic field due to differences in their masses. • The lighter the mass, the more they are deflected. • It also depends upon the no. of +ve charge an ion is carrying; the more +ve charge, the more it will be deflected.
  • 19. 19 Detection • The beam of ions passing through the mass analyzer is detected by a detector on the basis of the m/e ratio. • When an ion hits the metal box, the charge is neutralized by an electron jumping from the metal onto the ion. • Types of analyzers: • Magnetic sector mass analyzers • Double focussing analyzers • Quadrupole mass analysers • Time of Flight analyzers (TOF) • Ion trap analyzer • Ion cyclotron analyser
  • 20. 20 Applications of Mass Spectrometry (MS) • Environmental monitoring and analysis (soil, water, and air pollutants, water quality, etc.) • Geochemistry – age determination, soil, and rock composition, oil and gas surveying • Chemical and Petrochemical industry – Quality control • Identify structures of biomolecules, such as carbohydrates, nucleic acids • Sequence biopolymers such as proteins and oligosaccharides • Determination of the molecular mass of peptides, proteins, and oligonucleotides. • Monitoring gases in patients’ breath during surgery. • Identification of drug abuse and metabolites of drugs of abuse in blood, urine, and saliva. • Analyses of aerosol particles. • Determination of pesticides residues in food. •
  • 21. a)Mass spectrometry is quickly becoming the gold standard by which to identify protein sequences due to its ease of automation and extreme accuracy. c)The two most popular methods to identify protein sequences using Mass Spectrometry are Peptide Mass Fingerprinting and Tandem Mass Spectrometry. 21
  • 22. PEPTIDE MASS FINGERPRINTING 22 • Peptide mass fingerprinting (PMF), also known as protein fingerprinting, is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer such as MALDI-TOF or ESI-TOF. • The method was developed in 1993 by several groups independently. • The peptide masses are compared to either a database containing known protein sequences or even the genome. • This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. • The results are statistically analyzed to find the best match. • The advantage of this method is that only the masses of the peptides have to be known. A disadvantage is that the protein sequence has to be present in the database of interest.
  • 23. 23 • The peptides are then dissolved in a small amount of distilled water or further concentrated and purified and are ready for mass spectrometric analysis. • The digested protein can be analyzed with different types of mass spectrometers such as ESI-TOF or MALDI-TOF. • MALDI-TOF is often the preferred instrument because it allows a high sample throughput and several proteins can be analyzed in a single experiment. • The proteins are cut into several fragments using proteolytic enzymes such as trypsin, chymotrypsin or Glu-C. A typical sample:protease ratio is 50:1. • The proteolysis is typically carried out overnight and the resulting peptides are extracted with acetonitrile and dried under vacuum. • The mass spectrometric analysis produces a list of molecular weights which is often called a peak list. The peptide masses are compared to protein databases such as Swissprot, which contain protein sequence information. Software performs in silico digests on proteins in the database with the same enzyme (e.g. trypsin) used in the chemical cleavage reaction. The mass of these peptides is then calculated and compared to the peak list of measured masses.
  • 24. 24 • A small fraction of the peptide (usually 1 microliter or less) is pipetted onto a MALDI target and a chemical called a matrix is added to the peptide mix. • Common matrices are sinapinic acid, Alpha-Cyano-4-hydroxycinnamic acid, and 2,3- Dihydroxybenzoic acid. The matrix molecules are required for the desorption of the peptide molecules. • Matrix and peptide molecules co-crystallize on the MALDI target and are ready to be analyzed. • MALDI-MS sample preparation technique; dried droplet technique; The target is inserted into the vacuum chamber of the mass spectrometer and the desorption and ionisation of the polypeptide fragments is initiated by a pulsed laser beam which transfers high amounts of energy into the matrix molecules. • The energy transfer is sufficient to promote the ionisation and transition of matrix molecules and peptides from the solid phase into the gas phase. • The ions are accelerated in the electric field of the mass spectrometer and fly towards an ion detector where their arrival is detected as an electric signal.
  • 26. ANALYSIS OF C TERMINAL AMINOACIDS 26 • Fewer methods of analysis are available for the identification of C-terminal residue of polypeptides. Hydrazinolysis: • Proteins are treated with anhydrous hydrazine (H2N-NH2) at 100° C. • So, all the amino acids of polypeptide chain is reacted with hydrazine and converted into amino acid hydrazides. • But, the amino acid attached to carboxyl terminal is not reacted and remain as the free amino acid. • This free amino acid can be isolated and identified by chromatographic analysis.
  • 27. 27 LiAlH4 (Lithium aluminium hydride) based reduction of C-terminus: • Proteins are analysed by this method through the process of reduction. • Reduction of free α-amino group at the end of a peptide chain into an alcohol is performed by LiAlH4. • After the reduction process, subsequent acid hydrolysis of protein is carried out and free-amino acids with a single free alcohol are obtained. • This single free alcohol represented the C-terminal residue. Enzymatic analysis or carboxypeptidase-based analysis: • Most recent and popular method of analysis of C-terminus of protein is the enzymatic analysis or carboxypeptidase-based analysis. • The enzyme carboxypeptidase cut the amino acid residues from the C-terminus of a protein in a sequential way. • Free amino acids from C-terminus of a protein can be isolated and identified by chromatographic analysis.
  • 28. SIGNIFICANCE OF PROTEIN SEQUENCING 28 • Information of the sequence of a protein is very essential in explaining its mechanism of action. • The three-dimensional structure of proteins is explained by the information of amino acid sequence. • Amino acid sequence provides a relation between the genetic message in RNA and three- dimensional structure that performs a proteins biological function. • Amino acid sequence determination is an essential part of molecular pathology. • Alterations in amino acid sequence can produce abnormal function of protein and resulting in disease development, such as • sickle cell anemia & cystic fibrosis. • Evolutionary history is also determined by protein sequencing. • Protein resembles with another amino acid sequence only if they have a common ancestor. Therefore, molecular events in evolution can be traced from amino acid sequences.
  • 29. PROTEIN MICROARRAY 29 • A protein microarray (or protein chip) is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. • Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. • Probe molecules, typically labeled with a fluorescent dye, are added to the array. • Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. • Protein microarrays are rapid, automated, economical, and highly sensitive.
  • 30. • The proteins are arrayed onto a solid surface such as microscope slides, membranes, beads or microtitre plates. • The function of this surface is to provide a support onto which proteins can be immobilized, maximal binding properties, whilst maintaining the protein in its native conformation so that its binding ability is retained. • Microscope slides made of glass or silicon are a popular choice since they are compatible with the easily obtained robotic arrayers and laser scanners that have been developed for DNA microarray technology. • Nitrocellulose film slides are broadly accepted as the highest protein binding substrate for protein microarray applications. METHOD • The chosen solid surface is then covered with a coating that must serve the simultaneous functions of immobilising the protein, preventing its denaturation, orienting it in the appropriate direction so that its binding sites are accessible, and providing a hydrophilic environment in which the binding reaction can occur. • Immobilising agents include layers of aluminium or gold, hydrophilic polymers, and polyacrylamide gels, or treatment with amines, aldehyde or epoxy. • Thin-film technologies like physical vapour deposition (PVD) and chemical vapour deposition (CVD) are employed to apply the coating to the support surface. 30
  • 31. • In the most common type of protein array, robots place large numbers of proteins or their ligands onto a coated solid support in a pre- defined pattern. This is known as robotic contact printing or robotic spotting. • Another method is ink-jetting, non-contact method of dispersing the protein polymers onto the solid surface in the desired pattern. • Piezoelectric spotting is a similar to ink-jet printing. The printhead moves across the array, and at each spot uses electric stimulation to deliver the protein molecules onto the surface via tiny jets. This is also a non-contact process. • Photolithography is a fourth method of arraying the proteins onto the surface. Light is used in association with photomasks. • The most widely used method for detection is fluorescence labeling which is highly sensitive, safe and compatible with readily available microarray laser scanners. • Other labels can be used, such as affinity, photochemical or radioisotope tags. • These labels are attached to the probe itself and can interfere with the probe-target protein reaction. • Label free detection methods; surface plasmon resonance (SPR), carbon nanotubes, carbon nanowire sensors (where detection occurs via changes in conductance) and microelectromechanical system (MEMS) cantilevers.[ 31 An aqueous environment is essential at all stages to prevent protein denaturation. Therefore, sample buffers contain a high percent of glycerol (to lower the freezing point), and the humidity of the manufacturing environment is carefully regulated. Microwells have the dual advantage of providing an aqueous environment while preventing cross-contamination between samples.
  • 32. PROCEDURE 32 Selection of Proteins: • Choose a set of proteins relevant to the research question or experiment. These can be purified proteins, protein fragments, or even whole-cell lysates. Microarray Substrate Preparation: • Select a solid support for the microarray, such as a glass slide, membrane, or a specialized chip. • Clean the substrate to ensure a proper surface for protein immobilization. Immobilization of Proteins: • Choose an immobilization method based on the microarray substrate and experimental requirements. • Spotting: Use robotic spotters or printers to deposit small droplets of protein solutions onto the substrate. • In Situ Synthesis: Utilize techniques like photochemical reactions or inkjet printing to synthesize proteins directly on the substrate. • Surface Immobilization: Covalently attach proteins to the substrate through chemical reactions or physically adsorb them.
  • 33. 33 Blocking and Stabilization: • Block the remaining active sites on the substrate to prevent non-specific binding of proteins during subsequent steps. • Stabilize the immobilized proteins to maintain their activity and conformation. Sample Incubation: • Incubate the microarray with a biological sample containing proteins of interest (e.g., cell lysates, purified proteins, or serum). • Allow time for specific interactions to occur between the immobilized proteins and those in the sample. Washing: • Wash the microarray to remove unbound or nonspecifically bound proteins, reducing background noise. • Use buffer solutions with appropriate detergents and salts for efficient washing.
  • 34. 34 Detection Method: • Choose a suitable detection method based on the experimental goals and the nature of the proteins being studied. Fluorescence Detection: • Label proteins with fluorescent dyes or tags. • Use a fluorescence scanner to visualize and quantify signals. Chemiluminescence: • Utilize enzymatic reactions to produce light. • Image chemiluminescent signals using specialized equipment. Radioactive Labeling: • Label proteins with radioactive isotopes (less common due to safety concerns). • Use autoradiography for detection.
  • 35. 35 Data Acquisition: • Capture images or signals generated by the detection method. • Collect data on the intensity, location, and patterns of signals from the microarray. Data Analysis: • Analyze the data using bioinformatics tools and software. • Interpret the results, identify protein interactions, assess expression levels, or investigate post- translational modifications. Validation: • Validate the results using additional experimental techniques, such as immunoprecipitation, Western blotting, or other assays, to confirm the observed protein interactions. Interpretation and Conclusion: • Draw conclusions based on the analyzed data. • Discuss the biological significance of the findings and their implications for the research question.
  • 36. APPLICATIONS OF PROTEIN MICROARRAY 36 Disease Biomarker Discovery: • Objective: Identify protein biomarkers associated with diseases. • Application: Early detection, prognosis, and monitoring of diseases such as cancer, neurodegenerative disorders, and autoimmune diseases. Diagnostic Tools in Medicine: • Objective: Develop diagnostic assays for diseases. • Application: Protein microarrays can be used for clinical diagnostics, allowing for rapid and multiplexed detection of disease markers in patient samples. Drug Target Discovery and Validation: • Objective: Identify potential drug targets and validate their relevance. • Application: Accelerate drug discovery by profiling proteins to discover targets, assess drug interactions, and validate potential therapeutic candidates.
  • 37. 37 Pharmacogenomics: • Objective: Personalize drug treatments based on individual genetic variations. • Application: Identify patient-specific protein expression patterns to predict responses to drugs, optimize dosages, and minimize adverse effects. Protein-Protein Interaction Studies: • Objective: Investigate interactions between proteins. • Application: Understand cellular signaling pathways, protein complexes, and regulatory networks, providing insights into cellular functions. Functional Proteomics: • Objective: Study the function of proteins in a systematic manner. • Application: Characterize the roles of proteins in cellular processes, signaling cascades, and pathways to gain a comprehensive understanding of cellular function.
  • 38. 38 Autoimmune Disease Research: • Objective: Investigate autoantibody profiles and protein targets in autoimmune diseases. • Application: Uncover autoimmune disease mechanisms, identify autoantigens, and develop diagnostic tools for autoimmune conditions. Allergy Profiling: • Objective: Identify allergenic proteins and study allergic responses. • Application: Characterize allergic reactions by detecting specific IgE antibodies against allergens, aiding in allergy diagnosis and treatment. Vaccine Development: • Objective: Identify immunogenic proteins for vaccine candidates. • Application: Screen and identify potential vaccine antigens by assessing antibody responses and epitope mapping.
  • 39. 39 Pathogen Detection and Characterization: • Objective: Identify and characterize proteins from pathogens. • Application: Develop diagnostic tools for infectious diseases, study pathogen-host interactions, and identify potential vaccine targets. Stem Cell Research: • Objective: Study protein expression profiles during stem cell differentiation. • Application: Understand the molecular mechanisms involved in stem cell development and differentiation. Environmental Monitoring: • Objective: Detect and quantify proteins indicative of environmental conditions or pollution. • Application: Monitor environmental changes, assess pollution levels, and study the impact on living organisms.
  • 40. 40 Environmental Monitoring: • Objective: Detect and quantify proteins indicative of environmental conditions or pollution. • Application: Monitor environmental changes, assess pollution levels, and study the impact on living organisms. Neuroscience Research: • Objective: Investigate protein expression patterns in the nervous system. • Application: Study neurological disorders, neuronal development, and synaptic functions. Cell Signaling Pathway Analysis: • Objective: Explore signaling pathways and protein cascades. • Application: Investigate how proteins communicate within cells, helping to understand cellular responses to stimuli and external factors.
  • 41. ANALYTICAL PROTEIN MICROARRAY 41 • Analytical microarray are also known as capture arrays, uses a library of antibodies, aptamers or affibodies arrayed on the support surface. • These are used as capture molecules since each binds specifically to a particular protein. • The array is probed with a complex protein solution such as a cell lysate. • This type of microarray is especially useful in comparing protein expression in different solutions. • For instance the response of the cells to a particular factor can be identified by comparing the lysates of cells treated with specific substances or grons under certain conditions with the lysates of control cells. • Another application is in the identification and profiling of diseased tissues. Types of protein microarray
  • 42. FUNCTIONAL PROTEIN MICROARRAY 42 • Functional protein microarrays (also known as target protein arrays) are constructed by immobilizing large numbers of purified proteins and are used to identify protein-protein, protein-DNA, protein-RNA, protein-phospholipid and protein-small- molecule interactions, to assay enzymatic activity and to detect antibodies and demonstrate their specificity. • They differ from analytical arrays in that functional protein arrays are composed of arrays containing full-length functional proteins or protein domains. • These protein chips are used to study the biochemical activities of the entire proteome in a single experiment.
  • 43. GFP TAGGING 43 • Green Fluorescent Protein (GFP) was first isolated from the jelly fish, Aequorea victoria. • The protein has 238 amino acids, three of them (65 to 67) form a structure that emits visible green fluorescent light when exposed to UV light. • Biologists use GFP to study cells in embryos and fetuses during developmental processes and also as a marker protein. • GFP can attach to and mark another protein with fluorescence, enabling scientists to see the presence of the particular protein in an organic structure. • Using rDNA technology, scientists combine the gfp gene to another gene that produces a protein that they want to study, and then they insert the complex into a cell. • If the cell produces the green fluorescence, scientists infer that the cell expresses the target gene. • Moreover, scientists use GFP to label specific organelles, cells, tissues.
  • 44. 44 • As the Gfp gene is heritable, the descendants of labeled entities also exhibit green fluorescence. • The gene for GFP was successfully inserted into E.coli bacteria in 1994 and the Nobel Prize in Chemistry 2008 was jointly awarded to Osamu Shimomura, Martin Chalfie and Roger Y. Tsien for the discovery and development of GFPs. • GFP is generally non-toxic and can be expressed to high levels in different organisms with minor effects on their physiology. • When the gene for GFP is fused to the gene of a protein to be studied, the expressed protein of interest retains its normal activity. • Likewise, GFP retains its fluorescence, so that the location, movement and other activities of the studied protein can be followed by microscopic monitoring of the GFP fluorescence
  • 45. STRUCTURE OF GFP 45 • The structure includes primary structure of a chain of 238 amino acids, a secondary structure of hydrogen-bonded helices and pleated sheets and a tertiary barrel shaped structure capped with helices. • At the center - chromophore, an amino acid section responsible for the light emission. • Particularly useful for fluorescence microscopy as the chromophore is encased in the barrel shaped structure, avoiding solvents and allowing the protein to fluoresce in many conditions. • Unlike other proteins that require enzymes for the complex folding necessary for the required protein shape, this occurs automatically in GFP.
  • 46. REFERENCES 46 • eGyanKosh https://egyankosh.ac.in › unit-6PDF SEQUENCING AND ANALYSIS OF PROTEIN. • Future Science https://www.future-science.com › doi Protein Microarrays | BioTechniques • Mark Schena (2005). Protein Microarrays Jones & Bartlett Learning. pp. 47–. ISBN 978-0- 7637-3127-4 • Remington S. J. (2011). Green fluorescent protein: a perspective. Protein science : a publication of the Protein Society, 20(9), 1509–1519. https://doi.org/10.1002/pro.684 • UPV/EHU https://www.ehu.eus › papersPDF Peptide Sequencing by Edman Degradation. • Zou, Yawen, "Green Fluorescent Protein”. Embryo Project Encyclopedia ( 2014-06-11 ). ISSN: 1940-5030 https://hdl.handle.net/10776/7903