Transcriptome analysis; Proteomics Flashcards
Transcriptome analysis attempts to
catalog and quantify the total RNA content of a cell, tissue, or organism.
Transcriptome analysis reveals gene-expression profiles that, for the same genome, may
vary from cell to cell or from tissue type to tissue type
Transcriptome analysis provides insights into:
■ normal patterns of gene expression that are important for understanding how a cell or tissue type differentiates during development
■ how gene expression dictates and controls the physiology of differentiated cells
■ mechanisms of disease development that result from or cause gene-expression changes in cells.
For example, examining gene-expression profiles in a cancerous tumor can help diagnose tumor type, determine the likelihood of tumor metastasis (spreading), and develop the most effective treatment strategy.
Transcriptome analysis also called
transcriptomics or global analysis of gene expression
Transcriptome analysis studies the expression of genes in a genome
both qualitatively and quantitatively.
Qualitatively
- by identifying which genes are expressed or not expressed.
Quantitatively
- by measuring varying levels of expression for different genes.
For nearly two decades DNA microarray analysis has been widely used because it
enables researchers to analyse all of a sample’s expressed genes simultaneously.
A single microarray can have over 20,000 different spots of DNA, each containing
a unique sequence that serves as a probe* for a different gene.
Probe
need to have sequence information about the genes of interest
PCR-based methods - such as reverse transcription PCR (RT-PCR) and quantitative real-time PCR (qPCR) are useful because
of their ability to detect genes expressed at low levels.
Now, RNA-Seq has superseded arrays – totally dominate expression analysis.
Qualitative and quantitative for totally unknown samples.
Microarray Analysis
DNA Microarray Analysis – How does one interpret an experiment?
In this experiment a microarray was used to analyse gene expression patterns as a plant was infected with a pathogen – over a period of 290 min (4.8 hours). Assume it was an Arabidopsis plant and we used the Affymetrix gene chip (picture to the right). Looking more closely we see on the chip (picture on the left), specific clusters of genes being up- or down-regulated as the infection progresses. This gives us valuable information as to how Arabidopsis responds to a pathogen infection.
Microarrays are limited
As they can only study the expression of the genes with probes on the chip.
Direct RNA sequencing (RNA-seq) - called whole-transcriptome shotgun sequencing - allows for
quantitative analysis of all RNAs expressed in a particular tissue and, also provides actual sequence data.
Proteomics
the identification, characterisation, and quantitative analysis of proteomes
The Proteome is defined as
the complete set of proteins encoded by a given genome
Studying the proteome can yield valuable information about:
A protein’s structure and function
Posttranslational modifications
Protein-protein, protein-nucleic acid, and protein-metabolite interactions
Cellular localisation of proteins
Protein stability and aspects of translational and post-translational levels of gene-expression regulation
Relationships (shared domains, evolutionary history) to other proteins
Proteomes are larger than genomes
[Sequencing of mRNAs from human tissues found that over 95 % of protein-coding genes with more than one exon are alternatively spliced.]
Different transcriptional start sites
Alternative mRNA splicing
RNA editing
Post-translational modifications (> 100 mechanisms are known)
Human Proteome Map (HPM) – to cataloque the human proteome (2014)
About __________ of the Drosophila proteome has been well catalogued using proteomics.
two-thirds
Proteomics is also of clinical value because it
allows comparison of proteins in normal and diseased tissues, which can lead to the identification of proteins (or smallRNAs) as bio-markers for disease conditions.
Genes can have multiple transcription start sites that produce several different types of RNA transcripts.
Alternative splicing and editing of pre-mRNA molecules can generate dozens of different proteins from a single gene.
determined in large part by its gene-expression patterns
The specific protein content (or profile) of a cell - its transcriptome
However, a number of other factors affect the proteome profile of a cell.
To begin with, many proteins undergo co-translational or posttranslational modifications, such as cleavage of a signal sequence that targets a protein for an organelle pathway, a propeptide, or initiator methionine residues; linkage to carbohydrates and lipids; or the addition of chemical groups through methylation, acetylation, and phosphorylation; and other modifications. Over a hundred different mechanisms of posttranslational modification are known.
Early Proteomics - Two-dimensional gel electrophoresis (2DGE)steps
- Proteins are isolated from cells or tissues and first loaded onto a polyacrylamide tube gel and separated by isoelectric focusing, which causes proteins to migrate based on their electrical charge in a pH gradient.
- During isoelectric focusing, proteins migrate until they reach the location in the gel where their net charge is zero relative to the pH of the gel.
- In a second migration, perpendicular to the first, the proteins are separated by their molecular weight using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
- Proteins are identified by cutting spots out of a gel and sequencing short stretches of the amino acids that the spots contain.
- BLAST can then be used to search protein databases containing amino acid sequences of known proteins.
- However, because of alternative splicing or posttranslational modifications, peptide sequences may not always match easily and, the protein may have to be confirmed by another approach.
Two-dimensional gel electrophoresis (2DGE)
Mass spectrometry techniques analyze
ionized samples in gaseous form and measure the mass-to-charge (m/z) ratio of the different ions in a sample
Proteins analyzed by mass spectrometry generate m/z spectra that can be correlated with an
m/z database containing known protein sequences to discover the protein’s identity.
Some of the applications of Mass spectrophotometry (MS) are to:
Identify an unknown protein or proteins in a complex mix of proteins
Sequence peptides
Identify posttranslational modifications of proteins
Characterize multiprotein complexes.
One common MS approach is matrix-assisted laser desorption ionization (MALDI).
This approach is ideally suited for
identifying proteins and is widely used for proteomic analysis of tissue samples.
MS MALDI
MALDI employs an ultraviolet laser to heat, vaporize, and ionize peptide fragments. Released ions are then analyzed for mass; MALDI displays the m/z ratio of each ionized peptide as a series of peaks representative of the molecular masses of peptides in the mixture and their relative abundance.
Automated, high-throughput instruments can excise all spots froom a 2D gel for MALDI analysis.
MALDI produces a peptide “fingerprint”, characteristic of the protein being analysed.
Can be compared to MALDI-generated m/z spectra of known proteins.
MS MALDI
MS - Dinosaurs and birds, distant cousins?
In this example, tissue was harvested from the femur bones of a T-Rex fossil. The T. rex proteins extracted from the tissue showed cross-reactivity with antibodies to chicken collagen and could be digested by the protease, collagenase.
These results suggested that the T. rex protein samples contained collagen, a major matrix component of bone, ligaments, tendons, and skin.
To definitively identify the presence of collagen, peptides from the T. rex samples were analyzed by mass spectrometry.
The m/z spectra for one of the T. rex peptides was identified from a database of m/z spectra as corresponding to collagen.
