Lecture 3 Flashcards
1
Q
Name the phases of biomarker development.
A
- Experimental design
- Discovery
- Qualification
- Verification
- Validation and Clinical assay development
2
Q
What does experimental design consist of?
A
- This stage includes sample selection, collection, processing and storage.
- Biomarker discovery.
Read slides
3
Q
Proximal Fluids
A
- e.g. saliva, pancreatic juice
- Biofluid closer to or in direct contact with the site of disease.
- Proximal fluids may have several properties that make them attractive for biomarker discovery.
- Are local sinks for proteins or peptides secreted, shed or leaked from diseased tissue.
3
Q
Why is sample collection and storage important?
A
- To preserve sample integrity.
- Must have defined collection and storage protocols.
3
Q
What are the types of Tumor Markers
A
- Hormones -hCG (human chorionic gonadotrophin); calcitonin; gastrin; prolactin; growth hormone, etc.)
- Enzymes - (acid phosphatase; alkaline phos-phatase; PSA)
- Proteins & Glycoproteins - (CA 125; CA 15.3; CA 19.9, etc.)
- Oncofetal antigens - (CEA, AFP)
- Receptors (ER, PR, EGFR)
4
Q
Distal biofluids (blood)
A
- preferred material for a final diagnostic test
- They are distant from the site of disease.
- analytical challenges posed by the complexity and depth of the proteome
- protein biomarker discovery is further complicated by the low relative abundance expected for specific markers
5
Q
Why the recent optimism for biomarker discovery?
A
Due to new technologies and resources such as:
* Advanced bioinformatics
* Mass-spectrometry based profiling and identification
* Liquid Chromatography
* High-throughput techniques
6
Q
Diagnostic (screening) biomarker
A
- A marker that is used to detect and identify a given type of cancer in an individual.
- These markers are expected to have high specificity and sensitivity
- For example, the presence of Bence–Jones protein in urine remains one of the strongest diagnostic indicators of multiple myeloma.
- PSA (Prostate Specific Antigen)
6
Q
Prognostic biomarker
A
- Used once the disease status has been established.
- Predict the probable course of the disease including its recurrence
- Have an important influence on the aggressiveness of therapy.
- For example, HER2 amplification and/or overexpression is a marker of poor prognosis in breast cancer.
7
Q
Stratification (predictive) biomarker
A
- serves to predict the response to a drug before treatment is started.
- Classifies individuals as likely responders or non-responders to a particular treatment.
- Example - ER which is overexpressed in about 70% of breast cancer cases.
- ER-positive cancers are more likely to respond to anti-estrogen therapies such as tamoxifen
8
Q
What are the types of experimental Design Fractionation
A
- Non-Chromatographic Separations
- Chromatographic Separations (Instrument-based: Liquid Chromatography (LC))
Make the samples less complex
8
Q
Non-Chromatographic Seperations for Biofluids (Serum/plasma/saliva/urine)
A
A. Proteominer
B. Immunodepletion
C. Nanotraps
D. Albuminome
9
Q
Proteominer
A
- novel sample preparation tool used for the compression of the dynamic range of protein concentrations in complex biological samples.
- Decreases the dynamic range & dilute abundant and concentrate trace proteins.
- Provides a method for overcoming this challenge, allowing the exploration of the entire proteome.
10
Q
ProtemoMiner Protein Enrichment Kit
A
- The ProteoMiner protein enrichment kit provides columns and all necessary reagents for accessing low-abundance proteins in a variety of biological samples and is compatible with the majority of downstream proteomics applications.
11
Q
ProteoMiner Sequential Elution Kit
A
The ProteoMiner sequential elution kit is available for researchers who wish to elute their proteins into multiple fractions to detect additional proteins.
12
Q
Proteominer & SELDI-TOF
A
- Improves the peak counts in SELDI analysis.
13
Q
Proteominer- Key Benefits
A
- Decreases the amount of high-abundance proteins without immunodepletion; prevents the loss of proteins bound to high-abundance proteins, which are inadvertently lost with immunodepletion products
- Enriches and concentrates low-abundance proteins that cannot be detected through traditional methods
- Can be used to decrease the dynamic range of the protein concentration in a variety of samples and is not dependent on a predefined set of antibodies as are immunodepletion products
- Can be used for differential expression analysis
- Is compatible with current downstream protein analysis techniques
13
Q
Immunodepletion
A
Immunodepletion is a method for removing a target molecule from a mixture. Depletion typically begins by adding an antibody targeting the molecule of interest.
14
Q
What are the tools used for immunodepletion
A
- Multiple Affinity Removal LC Column - Human 14
- Multiple Affinity Removal Spin Cartridge – Hu 14
15
Q
Limitations of Immunodepletion
A
*The sample can be diluted during the elution step.
*Ever-deeper mining of the proteome requires an ever-expanding set of immunodepletion products.
*Batch to batch variation of antibodies
15
Q
Explain Nanotrap Technology
A
- Nanotrap is a carbon-based capture-particle that can be as small as 100nm, comprising a molecular sieve portion and an analyte binding portion.
- Nanotrap particles are synthesized with “bait” for specific proteins or molecules to be analyzed.
- Nanotrap particles are introduced to body fluid
- Nanotrap particles concentrate and preserve highly labile analytes.
16
Q
Nanotrap Key Benefits
A
- Enriches and concentrates low abundance proteins in complex biofluid samples.
- Does not utilize antibodies for immunodepletion or immunoprecipitation.
- Simultaneously harvests multiple low-abundance proteins from a single sample.
- Decreases amount of high-abundance proteins present in samples.
- Compatible with protein analysis techniques (CoomasieTM, silver staining, western blotting, mass spectrometry analysis).
- Prevents protein degradation during sample processing.
- Simple format and quick sample processing technique with best in market results.
17
Q
Albuminome
A
- Peptides and proteins (albumin-bound peptide and protein complex) that are bound to serum albumin, as well as the albumin molecule itself, can yield important data for disease diagnosis and management
- Study of Albuminome involves identifying and characterizing proteins that interact with serum albumin
- At least 35 different proteins are carried by albumin along with drugs circulating in the bloodstream
18
Q
Non-Chromatographic Separations for tissue/cells
A
A. Molecular weight fractionation
B. Fluorescence activated cell sorting
C. Laser capture microdissection
D. Organelle Isolation
19
Molecular Weight Fractionation
Use spin columns with specific molecular cut-off membranes
20
Fluorescence activated cell sorting (FACS)
* Seperates cell that are phenotypically different from each other.
* Quantifies the cells which express the proteins of interest.
## Footnote
Read slides 37-39
21
Laser Capture Microdissection
* Researchers use a low-energy laser beam and special transfer film to lift a desired cell out of the tissue section, leaving all unwanted cells behind.
* They can then collect all the proteins that were present in the selected cells, map the protein pattern, and store the information in a computer database.
22
What is a limitation of laser capture microdissection?
The limitation of tissue in size and quantity from patient.
23
What is oraganelle isolation - subcellular fractionation?
* Enrichment of organelles or cellular compartments –More comprehensive identification of associated proteins
* Important to determine organelle enrichment using other techniques such as western blotting using specific antibodies
24
Organelle Isolation -Methods used
Traditional
* 1. Differential centrifugation
* 2. Density-gradient centrifugation
* 3. Differential detergent fractionation
Recently developed
* 4. Free-flow electrophoresis
* 5. Immunoaffinity purification
25
Differential Centrifugation
* A traditional organelle isolation method.
* Operates via sequential centrifugation of the cell or tissue homogenate.
* Based on differences in size and density. (larger and denser organelles sediment at lower centrifugal forces)
* However, fractions prone to contamination with organelles with similar sedimentation velocities
26
Density-gradient centrifugation
* A traditional method of organelle isolation
* Separates organelles based on continuous and discontinuous gradients.
* Use of various media with different osmolarities, viscosities or densities
* Cell homogenate added to top of medium and centrifuged
## Footnote
Read slide 47 for more detail
27
Continuous Gradient
* For Density - gradient centrifugation which is a traditional method of organelle isolation
* Density increases linearly across the tube
* Equilibrium separation occurs, organelles distribute throughout the gradient at their isopycnic point
* Better resolution of organelles but time-consuming to prepare gradient
28
DIscontinous Gradient
– Gradient is divided into fixed portions consecutively
– Different organelles enriched at different interphases of the medium
29
Differential Detergent Fractionation
* A traditional method of organelle seperation
* Use of buffers of increasing stringency
* Separation of proteins in native state according to 4 compartments - cytosolic, membrane and membrane organelle localised, soluble and DNA-associated nuclear, cytokeletal proteins.
30
Free-Flow electrophoresis (FFE)
* Recently developed organelle isolation method.
* Organelle seperation based on their net global isoelectric charge or electrophoretic mobility.
* Purified organelles retain their intactness and functionality
* Has been used to separate peroxisomal membranes, mitochondria, secretoryvesicles, plasma membrane vesicles, peroxisomes.
31
Immunoaffinity Purification - Membrane Proteins
* One-third of total proteome thought to be associated with membrane
* Plasma membrane proteins
* Proteins in cell membrane
* Subcellular membrane proteins
* Isolation of membrane proteins with magnetic beads is new technology. It has the advantage of very fast, simple protocols but is relatively expensive.
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