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BI407: Tumor Biology > L3 > Flashcards

L3 Flashcards

1
Q

Phases of biomarker development

A
  • Experimental design
  • discovery
  • qualification
  • verification
  • validation and clinical assay development
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2
Q

Experimental design

A

sample selection, collection, processing and storage. Sample includes serum, plasma, saliva, urine etc

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3
Q

discovery involves?

A

identification of potential biomarkers. Using 2D-DIGE, LC-MS/MS, SELDI-TOF, protein arrays

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4
Q

Qualification involves

A

confirmation of differential abundance of potential biomarkers. Using LC-MS/MS, SELDI-TOF, high-throughput screening

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5
Q

Verification involves

A

assessing the specificity and selectivity of potential biomarkers. Using LC-MS/MS, SELDI-TOF, high throughput screening

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6
Q

Validation involves

A

establishing sensitivity and specificity; assay optimization using RIA/ELISA

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7
Q

Examples of tumor markers

A
  • hormones
  • enzymes
  • proteins and glycoproteins
  • oncofetal antigens
  • receptors
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8
Q

examples of tumor marker hormones

A

hCG(human chorionic gonadotrophin), calcitonin, gastrin, prolactin, growth hormone

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9
Q

examples of tumor marker enzymes

A

acid phosphatase, alkaline phosphatase

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10
Q

examples of tumor marker proteins and glycoproteins

A

CA 125, CA 15.3, CA 19.9

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11
Q

Examples of tumor marker oncofetal antigens

A

CEA, AFP

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12
Q

Examples of tumor marker receptors

A

ER, PR, EGFR

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13
Q

Difference between proximal and distal biofluids

A

Proximal biofluids are those obtained from the vicinity of the affected area, while distal biofluids are collected from elsewhere in the body, often blood.

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14
Q

which is preferred for a final diagnostic test

A

Distal biofluids because it can provide systemic information and is relatively easy to collect compared to other biofluids.

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15
Q

Which is more attractive for biomarker discovery

A

Proximal fluids

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16
Q

what causes challenges with proteome analysis in distal fluids

A

complexity and depth of the proteome

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17
Q

what causes challenges with identifying protein biomarkers in distal fluids

A

specific markers may be present in low abundance compared to other proteins in the sample. This low abundance can make it difficult to detect and accurately quantify the biomarkers amidst the background of other proteins.

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18
Q

examples of proximal fluids

A

saliva, pancreatic juice

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19
Q

Why are proximal fluids better for biomarker discovery

A

They are sinks for proteins that come from diseased tissue. As a result, these fluids are enriched with potential disease biomarkers

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20
Q

Specific protocols for sample collection and storage

A
  • specify what collection tubes to use
  • defined collection and storage protocols
  • define strict variables to preserve sample integrity
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21
Q

Resources that can be used for biomarker discovery and validation

A
  • advanced bioinformatics
  • mass-spectrometry-based profiling and identification
  • liquid chromatography
  • high-throughput technologies
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22
Q

Types of biomarkers

A
  • diagnostic
  • prognostic
  • stratification
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23
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

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24
Q

examples of biomarkers

A

Bence-Jones protein found in urine is a strong indicator of multiple myeloma, Prostrate specific antigen (PSA) is a biomarker for prostrate cancer

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25
Q

Prognostic biomarker

A

Is used once the disease status has been established. They predict the probable course of the disease including its recurrence, and they therefore have an important influence on the aggressiveness of therapy .

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26
Q

Example of prognostic biomarker

A

HER2 amplification and/or overexpression is a marker of poor prognosis in breast cancer.

27
Q

Stratification (predictive) biomarker

A

It predicts the response to a drug before treatment is started. It classifies individuals as likely responders or non-responders to a particular treatment. Eg ER-positive cancers are more likely to respond to anti-estrogen therapies such as tamoxifen

28
Q

Fractionation

A

a technique used to separate complex mixtures of molecules into smaller, more manageable fractions based on their properties. Makes the sample less complex

29
Q

These properties include:

A

molecular weight (MW), shape, charge, pI, hydrophobicity and affinity through specific biomolecular interactions.

30
Q

What are the most widely used fractionation approaches

A

chromatography and electrophoresis

31
Q

Methods of non-chromatographic separations for biofluids (serum, plasma, saliva, urine)

A

A. Proteominer
B. Immunodepletion
C. Nanotraps
D. Albuminome

32
Q

Proteominer purpose

A

for the compression of the dynamic range of protein concentrations in complex biological samples.

33
Q

Proteominer challenge

A

The presence of high-abundance proteins in complex biological samples (for example, albumin and IgG in serum or plasma) making the detection of medium- and low-abundance proteins extremely challenging.

34
Q

Proteominer solution

A

By selectively capturing and concentrating medium and low-abundance proteins, ProteoMiner effectively reduces the dynamic range of protein concentrations in the sample.

35
Q

After proteominer protein enrichment, what is used to analyze the proteins?

A
  • SELDI-TOF
  • SDS-PAGE
36
Q

Proteominer benefits

A
  • Decreases high-abundance proteins without immunodepletion, preventing loss of proteins bound to them
  • Enriches and concentrates low-abundance proteins undetectable through traditional methods
  • Reduces dynamic range of protein concentration in various samples, independent of predefined antibodies
    enables differential expression analysis
  • Compatible with current downstream protein analysis techniques
37
Q

Immunodepletion for fractionation

A

is a method for removing a target molecule from a mixture.
Depletion typically begins by adding an antibody targeting the molecule of interest

38
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

39
Q

Features of the patented ceres Nanotrap

A
  • carbon-based capture particle
  • can be as small as 100nm
  • contains a molecular sieve protion and an analyte binding protion
40
Q

Nanotrap technology 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

41
Q

What type of dye does Nanotrap technology use

A

organic reactive dyes used in cytology, allergy testing and fabric staining

42
Q

Methods of non-chromatographic separations for tissues and cells

A

A. Molecular weight fractionation
B. Fluorescence-activated cell sorting
C. Laser capture microdissection
D. Organelle Isolation

42
Q

What is the most abundant human protein

A

HSA which represents over 50% of total proteins circulating in our bloodstream

42
Q

Molecular weight fractionation

A

Use spin columns with specific molecular cut-off membranes

43
Q

Fluorescence activated cell sorting (FACS)

A
  • enables the separation of cells based on phenotypic differences, despite their identical DNA
  • allows researchers to precisely isolate cells expressing specific proteins of interest and quantify the amount of these proteins they express.
44
Q

Steps of FACS

A
  • Cell counting using laser (blue light) which can also be used to measure cell size
  • separation of cells by antibody tagging and collection of cells
  • cell sorting using an electrical charge
45
Q

Laser capture microdissection

A

used by researchers to precisely isolate specific cells from tissue sections for further analysis. Using a low-energy laser beam and special transfer film, researchers can selectively lift desired cells from the tissue section while leaving unwanted cells behind.

46
Q

Organelle isolation/ subcellular fractionation

A

involves the separation and enrichment of specific organelles or cellular compartments from a cell or tissue sample.

47
Q

How is organelle enrichment verified and why

A

using other techniques, such as western blotting with specific antibodies, to ensure the accuracy and reliability of the fractionation process and the subsequent identification of associated proteins.

48
Q

Traditional methods used for organelle isolation

A
  1. Differential centrifugation
  2. Density-gradient centrifugation
  3. Differential detergent fractionation
49
Q

Recently developed methods of organelle isolation

A
  1. Free-flow electrophoresis
  2. Immunoaffinity purification
50
Q
  1. Differential centrifugation
A
  • sequential centrifugation of cell or tissue homogenate
    separates nuclei, mitochondria, and lysosomes
  • Based on size and density differences
51
Q

which organelles sediment at lower centrifugal forces

A

larger and denser organelles

52
Q
  1. Density-gradient centrifugation
A

Separates organelles based on continuous and discontinuous gradients

53
Q

Continuous gradient

A
  • Density increases linearly across the tube
    – Equilibrium separation occurs, organelles distribute throughout the gradient at their isopycnicpoint
    – Better resolution of organelles but time-consuming to prepare gradient
54
Q

Discontinuous gradient

A
  • Gradient is divided into fixed portions consecutively
    – Different organelles enriched at different interphasesof the medium
55
Q
  1. Differential detergent fractionation
A

Use of buffers of increasing stringency
Separation of proteins in native state according to 4 compartments

56
Q

What are these four compartments

A
  • cytosolic
  • membrane and membrane organelle-localised soluble
  • DNA-associated nuclear
  • cytoskeletal proteins
57
Q

Free-flow electrophoresis (FFE)

A
  • Organelle separation based on their net global isoelectriccharge or electrophoreticmobility
    *
    Purified organelles retain their intactness and functionality
    *
    Has been used to separate peroxisomalmembranes, mitochondria, secretoryvesicles, plasma membrane vesicles, peroxisomes
58
Q

Immunoaffinity purification is mainly used for

A

membrane proteins

59
Q

Plasma membrane proteins

A

Located at cell surface, cell-cell interactions

60
Q

Proteins in cell membrane

A

Anchors for cytoskeletal proteins or ECM

61
Q
A

BI407: Tumor Biology (3 decks)

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