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Genomic and Proteomics > Chapter 1 Introduction to Proteomics > Flashcards

Chapter 1 Introduction to Proteomics Flashcards

1
Q

Classical single protein analysis

A

Gene -> mRNA -> Protein

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2
Q

Contemporary protein global analysis

A

Genome -> Transcriptome -> Proteomics

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3
Q

Proteome define

A

complete set of proteins expressed by cell, tissue or organism from DNA/mRNA

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4
Q

3 stimuli that proteome change with

A
  • Different temperatures
  • with or without addition of chemical
  • diseased vs healthy person
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5
Q

Proteomics

A

large scale, systematic analysis of proteins

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6
Q

2D PAGE to show example of proteome change

A

a representative 2DE gel of a normal tissue

Proteins that become down-regulated in tumor are shown with arrows and capital letters

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7
Q

Rationale for proteomics

A

Genomic DNA -> pre-mRNA -> mRNA -> protein -> functional protein

genomic DNA form genomics
mRNA form transcriptomics
functional proteins form proteomics

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8
Q

Pre-mRNA

A

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

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9
Q

2 goals of proteomics

A

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

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10
Q

Biomarkers

A

Disease biomarkers are substances that can be used as an indicator of the person’s biological state

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11
Q

2 Features of biomarkers

A
  • typically protein in nature

- detected in blood and body fluid

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12
Q

3 functions of biomarkers

A 
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

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13
Q

Ovarian Cancer

A

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

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14
Q

Personalized medicine

A
  • 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

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15
Q

Finding new drug targets (devising a drug to kill the skin cancer melanoma)

A

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

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16
Q

Rationale for proteomics

A

Transcriptional control - between genomic DNA and pre-mRNA
Translational control - between mRNA and protein
Post translational control - between protein and functional protein

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17
Q

PTMs

A

Proteins are post-translationally modified, resulting in a dynamic nature of proteins and proteomics

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18
Q

3 types of PTM

A

Glycosylation
Phosphorylation
Disulphide bonding

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19
Q

Extent and modification of PTMs

A

individual proteins
regulatory mechanisms within the cell
environmental factors

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20
Q

Proteomics and PTM

A

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

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21
Q

A single gene can produce many different mRNAs and protein

A

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

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22
Q

3 ways PTM affect protein properties

A
  • same protein backbone
    1. Biochemical properties (binding)
    eg. Disulphide bonds promote dimer multimer formation
  1. Chemical properties
    eg. Phosphorylation and glycosylation after change and pI of proteins
  2. Physical Properties (molecular weight)
    e. g. Glycosylation can alter molecular weights of proteins
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23
Q

Changes in physical/chemical properties are detected using electrophoretic means such as western blot

A

pI is the isoelectric point

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24
Q

Glycosylation

A
  • 50% glycosylated
  • Addition of sugar chains (oligosaccharides or glycan) to proteins to proteins during and after synthesis
  • Different extent of glycosylation results in heterogeneity
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25
Q

Another name for sugar chains addition

A

Moieties

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26
Q

Heterogeneity

A

The quality or state of being diverse in character or content.

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27
Q

Glycoproteomics

A

Identification, cataloging and characterization of glycoproteins

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28
Q

how does glycosylation affect the proteins

A

Increased solubility, bioactivity and circulation time in vivo

Bioactivity - required for proteins to fold properly
Stability - prevent proteases gaining access to protein surface

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29
Q

3 types of glycosylation

A

3 major types

  • N linked (N-glycan)
  • O linked (O-glycan)
  • Addition of GP (glycophosphatidylinositol) anchor
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30
Q

N linked glycan

A

sugars attached to a peptide chain through the asparagine residues

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31
Q

O linked glycan

A

sugars attached to a peptide chain through hydroxyl group of serine or threonine residues

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32
Q

Synthesis of N-linked glycan

A
  1. 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.
  2. Glycan transfer
    The glycan is then transferred to the nascent, growing polypeptide.
  3. Trimming and processing
    Sugars are trimmed off, and the polypeptide is then folded before being moved to the Golgi complex.
  4. Further trimming
    The glycoprotein goes through a series of further modifications
  5. Terminal glycosylation
    Ending with the capping of the oligosaccharide branches with sialic acid and fucose

Note: only occurs in eukaryotes

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33
Q

Synthesis of N linked glycan summary

A

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

34
Q

End result of N linked glycan

A

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

35
Q

Sialic acid

A

N-acetylneuraminic acid

widely distributed in animal tissues, mostly in glycoproteins

36
Q

Sialic acid

A

Generic term for N or O-substituted derivatives of neuraminic acid, a monosaccharide with 9-carbon backbone

37
Q

Part of the glycoproteins crosses the membrane

A

There are also parts on the cytoplasmic and extracellular sides attached to many different proteins on cell surface

38
Q

Cell receptor is sialic acid

A

Sialic acid linked to glycoproteins and gangliosides is used by many viruses as a receptor for cell entry.

39
Q

The spheres are sugars that are attached to many proteins

A

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

40
Q

Neuramidase activity

A

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

41
Q

Neuraminidase inhibitors

A

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

42
Q

Hemagglutinin

A

Glycoproteins which cause red blood cells (RBCs) to agglutinate or clump together

43
Q

2 drug names of neuraminidase

A

Tamiflu or Oseltamivir -> mimic salic acid

They bind to the active site of Neuraminidase

44
Q

Phosphorylation

A

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

45
Q

Phosphorylation, Ubiquitous form of PTM

A

Phosphorylation is the important form of regulatory modification in both prokaryotes and eukaryotes

It controls signal transduction, gene expression and regulation of cell division

46
Q

Significance of phosphorylation

A

In humans, abnormal phosphorylation is often associated with cancer

47
Q

3 amino acids associated with eukaryotes

A

In eukaryote – serine, threonine, tyrosine

48
Q

3 amino acids associated with prokaryotes

A

In bacteria – aspartic acid, glutamic acid and histidine

49
Q

Protein kinases

A

Enzymes that phosphorylate proteins

50
Q

Phosphatases

A

Enzymes that remove phosphate group

51
Q

Methods to determine if protein is phosphorylated

A
  • Divide protein into 2 tubes
  • Treat one with alkaline phosphatase
  • Run both on SDS PAGE/ Western blot
  • Difference in distance on gel
52
Q

Disulphide bonding

A

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

53
Q

Example of disulphide bonding insulin

A

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

54
Q

General workflow for proteomic analysis

A

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

55
Q

Sample preparation

A
  • 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
56
Q

Sample separation

A
  • 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)
57
Q

2 types of sample separation

A

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

58
Q

Sample separation by IEF

A

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

59
Q

Isoelectric point

A

pH at which proteins has no net charges

60
Q

How is isoelectric point affected by PTM

A

Glycosylation and phosphorylation affect the isoelectric point of proteins

61
Q

Sialic acid

A

negatively charged under high pH environment

can be separated using IEF

62
Q

Phosphate group

A

The phosphate group is negatively charged and can be separated using IEF

63
Q

IEF methods

A

the protein mixture is separated into different proteins at different pH

64
Q

Application of IEF

A

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

65
Q

EPO

A

protein with attached sugar (glycoprotein) produced in our kidney – misused as performance enhancing drug

66
Q

Released into bloodstream

A

bind to receptor in bone marrow
thus stimulate production of red blood cells
which increase blood oxygen carrying capacity

67
Q

Danger of recombinant human erythropoietin

A

Dehydration can link to the thickening of blood, increased viscosity

68
Q

Detection of changes in glycosylation by IEF - HuEPO and rHuEPO

A

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.

69
Q

Sample preparation by SDS-PAGE

A

SDS Polyacrylamide gel electrophoresis (SDS PAGE) separates proteins on the basis of their molecular weight

Glycosylation and phosphorylation affect the molecular weight of proteins

70
Q

SDS-PAGE 2nd dimension

A

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

71
Q

Detection of changes by phosphorylation

A

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

72
Q

1D PAGE to detect phosphorylation

A

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.

73
Q

Detection of changes in phosphorylation

A

80Da is not significant on SDS-PAGE
pi change appears on IEF

Typically no change in MW unless there is a change in conformation

74
Q

Visualization of phosphorylation by staining

A

coomassie staining
sliver staining
high MW - low MW

75
Q

Comparative analysis of spot intensity

A

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

76
Q

Comparative analysis of spot intensity

A

(Identification of co-expressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues

77
Q

Preparation of sample for mass spectrometry

- In-gel digestion

A

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

78
Q

In gel digestion process summary

A

1 - destaining
2 - reduction and alkylation
3 - in-gel digestion – enzyme treatment to cut protein into smaller fragments (peptides)
4 - extraction

79
Q

Mass spectrometry

A

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

80
Q

Mass spectrophotometry analysis

A

Ionisation
Target, matrix analysis, laser, extractionand electron optics
Flight path (heavier ions to lighter ions) via acceleration and arrangement
Detection

81
Q

Example of MS spectrum profile

A

x axis is mass m/-z
y axis is intensity
4700 MS?MS precursor 1016.47 Spec #1 => NF0.7 (BP = 73.3.8358)

82
Q

Summary

A

Understanding proteomics

Proteomes are dynamic

Biomarkers

Post Translational Modifications of proteins
Glycosylation, Phosphorylation, Disulphide bonding

Applications of proteomics

Genomic and Proteomics (9 decks)

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