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Why?
The number of found genes(ORFs) in human genome is much less than the number of expressed proteins, and therefore, more information is needed about the “working units” of the cells
General flow for proteomics analysis
Two-dimensional gel electrophoresis (2DE)
1st dimension
separation based on
the pI of proteins
• 2nd dimension
separation based on
the molecular weight
of proteins
• Several
visualization/detection
possibilities
Gel-based proteomics
• “Older”approach to screen the protein expression at the large scale
• The typical flow of gel-based proteomics (2DE & MS)
– Sample preparation
– First-dimension isoelectric focusing (IEF)
– Second-dimension SDS-PAGE
– Visualization & evaluation
– Expression analysis
– Protein identification by MS
MS driven techniques
MS identification of proteins after quantitative analysis by 2DE
– Peptide mass fingerprinting (Maldi MS)
– Sequence based identification (MS/MS)
• Identification and quantitation using MS
– Labeling samples (e.g. ICAT, iTRAQ) for quantitative analysis
– Identification of the post-translational
modifications
Generation of peptides for MS analysis
• Proteins digestion
– Typically by trypsin; cleaves on the C-side of Arg
and Lys i.e. generates peptides having R or K at
the C-terminus
– Also other cleaving enzymes e.g. clostripain,
endopeptidase Lys-C
• Peptide analysis
– Total mass of a peptide
– Peptide fragmentation (mass of single amino acids)
Maldi-ToF MS
Sample is cocrystallized with matrix and irradiated with laser, which leads to ionization of the peptides.
The time-of-flight of the peptides is measured, which allows the determination of the peptide masses.
Blotting tech.
A wide variety of techniques exist in the molecular laboratory.
Knowledge of the targets and appropriate techniques to detect them are an integral part of molecular methods in the clinical laboratory.
The following slides provide a review of targets and techniques used to detect the targets.
Blotting
All techniques use electrophoresis to separate.
Difference in techniques lies in the target
Four applications
Western
Southern
Northern
Southwestern
Western Blot
A technique used to identify and locate proteins based on their ability to bind to specific antibodies.
Detect protein of interest from a mixture of a great number of proteins.
Gives information about size of protein in comparison to size marker or ladder.
Similar in principle to ELISA, but is more specific.
Western Blot
Procedure
Separate proteins by SDS-PAGE
Transfer proteins onto membrane
Add primary antibody to protein of interest
Add secondary antibody, specific for primary antibody, attached to an enzyme
Add substrate to visualize bands.
Western Blot
Southern Blot
Southern blot hybridization is one of the most commonly used molecular techniques to detect specific DNA sequences using labeled probes.
Four steps:
DNA extraction
Electrophoresis to separate
Transfer to membrane
Use labeled probes, which will hybridize to specific sequence, to identify sequence of interest
Southern Blot
Southern Blot
Northern Blot
Used to study gene expression.
Similar to Western Blot but MAJOR difference is that RNA is analyzed.
Gels may be run on either agarose or denaturing polyacrylamide, the latter being preferable for smaller RNA fragments.
Formaldahyde is added to gel and acts as a denaturant to agarose.
For polyacrylamide, urea is the denaturant.
Not used much for diagnostic, mainly used in research.
Northern Blot
Three types of RNA: tRNA, rRNA and mRNA
Northern blot isolates and hybridizes mRNA
Procedure
mRNA extracted from cells and purified
Separate with electrophoresis
Transfer onto membrane
Use labelled probes to identify mRNA of interest
Northern Blot
Northern Blot
Southwestern Blot
Combines features of Southern and Western blotting techniques.
For rapid characterization of both DNA binding proteins and their specific sites on genomic DNA.
Involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA) by their ability to bind to a specific oligonucleotide probes.
Identification of protein factors that bind to genes to turn them on or off is therefore important in investigating gene functions.
Primary use is for research, not clinical applications.
Southwestern Blot
Procedure
Separate proteins using SDS-PAGE
Renatured by removing SDS in presence of urea
Transfer to membrane
Genomic DNA of interest is digested by restriction enzymes, labeled and added to separated proteins.
Comparison of Blotting Methods
VNTR
Rectangle blocks represent repeated DNA sequences at a particular VNTR location
Repeats are tandem – clustered together and oriented in same direction
Repeats can be removed or added leading to alleles with different numbers of repeats.
VNTR
VNTR blocks can be extracted with restriction enzymes and analyzed by RFLP or amplified by PCR and size determined by electrophoresis
The picture below illustrates VNTR allelic length variation among 6 individuals.
VNTR
Important source of RFLP genetic markers used in linkage analysis (mapping) of genomes.
Has become essential to forensic crime investigation.
May use PCR.
Size determined by gel electrophoresis and Southern blotting to produce pattern of bands unique to each individual.
The likelihood of 2 unrelated individuals having same allelic pattern extremely improbable.
VNTR also being used to study genetic diversity and breeding patterns in animals.
VNTR – Clinical Applications
Microbiology
VNTR typing as the next gold standard in the molecular epidemiology of tuberculosis
Genotyping for early diagnosis of M. tuberculosis super-infection or mixed infection.
DNA fingerprinting
DNA Fingerprinting
• VNTR analysis = Variable Number Tandem Repeat
• Very short tandem repeats (2-60 bp) minisatellites
• Highly variable in number between individuals.
• Cutting at restriction sites outside repeats gives a fragment of variable size.
• Use probe containing the repeated sequence.
• Will detect many arrays.
• Pattern of bands is different for each individual like a fingerprint.
MODERN DNA SEQUENCING
What & Why?
“Sequencing” means finding the order of nucleotides on a piece of DNA .
Nucleotide order determines Amino acid order, and by extension, protein structure and function (proteomics)
An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal
What & Why, Ctd.
Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment
DNA technology is also extended to environmental, agricultural and forensic applications
The Sanger Technique
Uses dideoxynucleotides (dideoxyadenine, dideoxyguanine, etc)
These are molecules that resemble normal nucleotides but lack the normal -OH group.
Because they lack the -OH (which allows nucleotides to join a growing DNA strand), replication stops.
The Sanger method requires
Multiple copies of single stranded template DNA
A suitable primer (a small piece of DNA that can pair with the template DNA to act as a starting point for replication)
DNA polymerase (an enzyme that copies DNA, adding new nucleotides to the 3’ end of the template
A ‘pool’ of normal nucleotides
A small proportion of dideoxynucleotides labeled in some way ( radioactively or with fluorescent dyes)
The template DNA pieces are replicated, incorporating normal nucleotides, but occasionally and at random dideoxy (DD) nucleotides are taken up.
This stops replication on that piece of DNA
The result is a mix of DNA lengths, each ending with a particular labeled DDnucleotide.
Because the different lengths ‘travel’ at different rates during electrophoresis, their order can be determined.
Originally four separate sets of DNA, primer and a single different DD nucleotide were produced and run on a gel.
Modern technology allows all the DNA, primers, etc to be mixed and the fluorescent labeled DDnucleotide ‘ends’ of different lengths can be ‘read’ by a laser.
Additionally, the gel slab has been replaced by polymer filled capillary tubes in modern equipment
PROTEOMICS DATABASES
Question: Find the theoretical MW and PI of a known protein and The position on 2D gel
--Beta actin from human
Browse information and view structure.
Open an internet browser, go to http://tw.expasy
Select SWISS-PROT and TrEMBL database
Type:alkaline phosphatase E. coli (P00634 )
“beta actin human” Will result in information about protein precursor (P60709)
Select Compute pI/Mw
Select residues 2-375 (this is the protein without the signal peptide), This will provide information about the protein’s average MW