Chapter 1 Introduction to Proteomics Flashcards
Classical single protein analysis
Gene -> mRNA -> Protein
Contemporary protein global analysis
Genome -> Transcriptome -> Proteomics
Proteome define
complete set of proteins expressed by cell, tissue or organism from DNA/mRNA
3 stimuli that proteome change with
- Different temperatures
- with or without addition of chemical
- diseased vs healthy person
Proteomics
large scale, systematic analysis of proteins
2D PAGE to show example of proteome change
a representative 2DE gel of a normal tissue
Proteins that become down-regulated in tumor are shown with arrows and capital letters
Rationale for proteomics
Genomic DNA -> pre-mRNA -> mRNA -> protein -> functional protein
genomic DNA form genomics
mRNA form transcriptomics
functional proteins form proteomics
Pre-mRNA
exists only briefly before it is fully processed into mRNA
2 different types of organisms
- introns and exons
Exons are retained in the final mRNA, introns are removed by splicing
2 goals of proteomics
1) Obtain a global, integrated view of the biology of an organism/tissue or cells by studying proteins together rather than individually
2) Quantitative changes in protein expression levels and apply the information to drug discovery and therapy
Monitor the properties of the ENTIRE COMPLEMENTS OF PROTEINS from a given cell or organism, and to determine how these properties change in response to various physiological states, such as signaling ligands, cell cycle, and disease
Biomarkers
Disease biomarkers are substances that can be used as an indicator of the person’s biological state
2 Features of biomarkers
- typically protein in nature
- detected in blood and body fluid
3 functions of biomarkers
Differentiate state of disease in patients - search for cure for disease Gauge level of response to therapy - simplify prognosis Gauge level of drug administered - customize dosage
Biological features that correspond to a particular physiological data
Ovarian Cancer
4th most common cancer in women
Most women diagnosed at late clinical stage, with less than 30% survival in 5-year period
If detected at stage 1 , more than 90% survival rate for patients in 5 year period
Proteomics used to find biomarkers to diagnose stage 1 ovarian cancer with >94% accuracy vs current method of 35% accuracy
Personalized medicine
- Proteome of a human differ between and within populations such as metabolic enzymes
- Metabolism can influence drug efficacy and toxicity
- > poor metabolizes
- > ultra rapid metabolizers
Personalized medicine can help to increase benefit to risk ratio
Finding new drug targets (devising a drug to kill the skin cancer melanoma)
Melanoma extract out cancer tissue sample, 2D-PAGE - > overproduced protein identified from the gel -> microwell plate -> protein is isolated and crystallized -> x-ray crystallography reveals the proteins structures -> Drugs can be designed to block the proteins activity
Rationale for proteomics
Transcriptional control - between genomic DNA and pre-mRNA
Translational control - between mRNA and protein
Post translational control - between protein and functional protein
PTMs
Proteins are post-translationally modified, resulting in a dynamic nature of proteins and proteomics
3 types of PTM
Glycosylation
Phosphorylation
Disulphide bonding
Extent and modification of PTMs
individual proteins
regulatory mechanisms within the cell
environmental factors
Proteomics and PTM
50-90% of all proteins are PTM
Consequently, many proteins are present in multiple form
The type of PTM results in different types of proteomics
A single gene can produce many different mRNAs and protein
PTM increase the complexity of proteome significantly, especially in eukaryotes
Every protein can be modified in hundreds of different ways
Many PTMs are still being discovered when individual protein, complexes and pathways are being studied
3 ways PTM affect protein properties
- same protein backbone
1. Biochemical properties (binding)
eg. Disulphide bonds promote dimer multimer formation
- Chemical properties
eg. Phosphorylation and glycosylation after change and pI of proteins - Physical Properties (molecular weight)
e. g. Glycosylation can alter molecular weights of proteins
Changes in physical/chemical properties are detected using electrophoretic means such as western blot
pI is the isoelectric point
Glycosylation
- 50% glycosylated
- Addition of sugar chains (oligosaccharides or glycan) to proteins to proteins during and after synthesis
- Different extent of glycosylation results in heterogeneity
Another name for sugar chains addition
Moieties
Heterogeneity
The quality or state of being diverse in character or content.
Glycoproteomics
Identification, cataloging and characterization of glycoproteins
how does glycosylation affect the proteins
Increased solubility, bioactivity and circulation time in vivo
Bioactivity - required for proteins to fold properly
Stability - prevent proteases gaining access to protein surface
3 types of glycosylation
3 major types
- N linked (N-glycan)
- O linked (O-glycan)
- Addition of GP (glycophosphatidylinositol) anchor
N linked glycan
sugars attached to a peptide chain through the asparagine residues
O linked glycan
sugars attached to a peptide chain through hydroxyl group of serine or threonine residues
Synthesis of N-linked glycan
- Synthesis of lipid-linked precursor
First, sugars are linked onto a lipid precursor (in the cytosol), which is then flipped over into the lumen of the endoplasmic reticulum (ER) and the core oligosaccharide is finished. - Glycan transfer
The glycan is then transferred to the nascent, growing polypeptide. - Trimming and processing
Sugars are trimmed off, and the polypeptide is then folded before being moved to the Golgi complex. - Further trimming
The glycoprotein goes through a series of further modifications - Terminal glycosylation
Ending with the capping of the oligosaccharide branches with sialic acid and fucose
Note: only occurs in eukaryotes
Synthesis of N linked glycan summary
Begin with attachment of a branched 14- residues oligosaccharide – the core glycans – occurs in the ER because enzyme is localized in ER membrane
Core glycan is then trimmed by glycosidase
Partially glycosylated protein moved to the Golgi apparatus
Further modification takes place – substitution of certain core glycans residues and elaboration of glycan chains
note: only occurs in eukaryotes
End result of N linked glycan
More than 30 different types of sugar molecule can be added – structure of chains can vary significantly
Process of elaboration produced 3 major types of glycan structure – high mannose, hybrid, complex types
Sialic acid
N-acetylneuraminic acid
widely distributed in animal tissues, mostly in glycoproteins
Sialic acid
Generic term for N or O-substituted derivatives of neuraminic acid, a monosaccharide with 9-carbon backbone
Part of the glycoproteins crosses the membrane
There are also parts on the cytoplasmic and extracellular sides attached to many different proteins on cell surface
Cell receptor is sialic acid
Sialic acid linked to glycoproteins and gangliosides is used by many viruses as a receptor for cell entry.
The spheres are sugars that are attached to many proteins
Sialic acid is always the last sugar in a chain that is attached to a protein
The sialic acid is outside of the cell and acts as a receptor
Neuramidase activity
They are enzymes that cleave sialic acid groups from glycoproteins and viral glycoproteins
Budding virus in host cell, receptor containing sialic acid.
Neuraminidase cleave receptors, allowing the release of new virions, continued viral replication
Neuraminidase inhibitors
Antiviral agents that inhibit influenza viral neuraminidase activity and are of major importance in the control of influenza
They ensure no virions are released from the cell receptors, thus halting viral replication
Hemagglutinin
Glycoproteins which cause red blood cells (RBCs) to agglutinate or clump together
2 drug names of neuraminidase
Tamiflu or Oseltamivir -> mimic salic acid
They bind to the active site of Neuraminidase
Phosphorylation
Addition of phosphate groups to proteins (phosphoproteins)
Amino acids involve serine, threonine and tyrosine
Changes activity of proteins in a reversible manner “on” and “off” states eg.p53 phosphorylation
Phosphoproteomics - identification, cataloging and characterization of phosphoproteins
Phosphorylation, Ubiquitous form of PTM
Phosphorylation is the important form of regulatory modification in both prokaryotes and eukaryotes
It controls signal transduction, gene expression and regulation of cell division
Significance of phosphorylation
In humans, abnormal phosphorylation is often associated with cancer
3 amino acids associated with eukaryotes
In eukaryote – serine, threonine, tyrosine
3 amino acids associated with prokaryotes
In bacteria – aspartic acid, glutamic acid and histidine
Protein kinases
Enzymes that phosphorylate proteins
Phosphatases
Enzymes that remove phosphate group
Methods to determine if protein is phosphorylated
- Divide protein into 2 tubes
- Treat one with alkaline phosphatase
- Run both on SDS PAGE/ Western blot
- Difference in distance on gel
Disulphide bonding
covalent bond derived by the coupling of 2 thiol group
Cysteine residues in the protein backbone can lead to form bonds
Redox reactions catalyzed by enzymes, specifically thiol oxidoreductases
formation of inter and intra molecular bonds
results in dimers/multimers and folding of proteins
Example of disulphide bonding insulin
pre-insulin is a precursor to insulin
It is synthesized in the ER where it is folded and its disulfide bonds are oxidized
it is then transported to the golgi apparatus where it is packaged into secretory vesicles, then processed by a series of proteases to form mature insulin
General workflow for proteomic analysis
Sample -> Protein mixture -> Peptides -> MS data -> Protein identification
Between sample and protein mixture is sample preparation
Between protein mixture and peptides is samples separation and visualization, comparative analysis, digestion
Between peptides and MS data is mass spectrometry
Between MS data and protein identification is database search
Sample preparation
- to break open tissue/cells to release cellular contents (cell disruption)
- prefractionation such as use of chromatography may be carried out to enrich proteins of
1. certain cellular organelles/compartments
2. certain classes of proteins (glycoproteins) - Improve resolution of proteins in subsequent steps by reducing protein complexes into smaller components
1. reducing protein complexes into smaller components
2. breaking apart protein structure
Sample separation
- resolves protein mixture into individual proteins or small groups of proteins
- allow comparison of differences in protein levels between 2 samples (software)
- limit to a smaller subset of proteins for further analysis
- 2D polyacrylamide gel electrophoresis (2D-PAGE)
2 types of sample separation
small or large scale
methods range from fully selective (affinity based) to fully on-selective
regardless of methods, important to remember to exploit physical and chemical difference between proteins and cause them to behave different in particular environment
Sample separation by IEF
IEF is isoelectric focusing which separates proteins based on their isoelectric point
Acid base properties of amino acids are affected by environmental pH, protein will have net positive charge
Isoelectric point
pH at which proteins has no net charges
How is isoelectric point affected by PTM
Glycosylation and phosphorylation affect the isoelectric point of proteins
Sialic acid
negatively charged under high pH environment
can be separated using IEF
Phosphate group
The phosphate group is negatively charged and can be separated using IEF
IEF methods
the protein mixture is separated into different proteins at different pH
Application of IEF
Recombinant human erythropoietin (rHuEPO)
Glycoprotein (165 amino acids and 3 N-linked and 1 O-linked glycans)
Enhances athletic performance by increasing the number of erythrocytes
EPO
protein with attached sugar (glycoprotein) produced in our kidney – misused as performance enhancing drug
Released into bloodstream
bind to receptor in bone marrow
thus stimulate production of red blood cells
which increase blood oxygen carrying capacity
Danger of recombinant human erythropoietin
Dehydration can link to the thickening of blood, increased viscosity
Detection of changes in glycosylation by IEF - HuEPO and rHuEPO
The five lanes containing markers (lanes S) were spotted with 2 fmoles each of rHuEPO and darbepoetin.
Lanes QCP and QCN represent urines from individuals known to be receiving rHuEPO and not to be receiving rHuEPO, respectively.
The lanes in section A were obtained from a placebo-treated individual on post-administration days 2, 3, and 4.
The lanes in sections B and C were obtained from epoetin alfa-treated individuals on days 2, 3, 4, and 7.
Sample preparation by SDS-PAGE
SDS Polyacrylamide gel electrophoresis (SDS PAGE) separates proteins on the basis of their molecular weight
Glycosylation and phosphorylation affect the molecular weight of proteins
SDS-PAGE 2nd dimension
low pH - high pH for IEF (2nd dimension)
This the x axis at the top
High MW to low MW
This the Y axis from top to bottom
Detection of changes by phosphorylation
80Da is not significant on SDS-PAGE/Western Blot
Mobility shift due to change in protein conformation, even under denaturing conditions
80Da change can be picked up by Mass Spectrometry
1D PAGE to detect phosphorylation
In some very specific cases, the detection of the phosphorylation as a shift in the protein’s electrophoretic mobility is possible on simple 1-dimensional SDS-PAGE gels, as it’s described for instance for a transcriptional coactivator by Kovacs et al.
Strong phosphorylation-related conformational changes (that persist in detergent-containing solutions) are thought to underlie this phenomenon.
Detection of changes in phosphorylation
80Da is not significant on SDS-PAGE
pi change appears on IEF
Typically no change in MW unless there is a change in conformation
Visualization of phosphorylation by staining
coomassie staining
sliver staining
high MW - low MW
Comparative analysis of spot intensity
Use of software to compare differences in proteome across different samples
Spot intensity level of protein expression
Usually spots with large differences in intensity are chosen for further study
Labor-intensive process
Comparative analysis of spot intensity
(Identification of co-expressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues
Preparation of sample for mass spectrometry
- In-gel digestion
Recover protein(s) of interest from gel for further identification
Break protein(s) into smaller peptides for ease of analysis
Automated machines available to speed up process
In gel digestion process summary
1 - destaining
2 - reduction and alkylation
3 - in-gel digestion – enzyme treatment to cut protein into smaller fragments (peptides)
4 - extraction
Mass spectrometry
Provides accurate molecular mass measurements of proteins or digested peptides
The data from these mass measurements can be referenced against databases to obtain:
identity of target proteins/digested peptides
sequence of target proteins/digested peptides
Modern machines allow detection at very small quantities of samples
Mass spectrophotometry analysis
Ionisation
Target, matrix analysis, laser, extractionand electron optics
Flight path (heavier ions to lighter ions) via acceleration and arrangement
Detection
Example of MS spectrum profile
x axis is mass m/-z
y axis is intensity
4700 MS?MS precursor 1016.47 Spec #1 => NF0.7 (BP = 73.3.8358)
Summary
Understanding proteomics
Proteomes are dynamic
Biomarkers
Post Translational Modifications of proteins
Glycosylation, Phosphorylation, Disulphide bonding
Applications of proteomics
