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URI > Genomics and proteomics > Flashcards

Genomics and proteomics Flashcards

1
Q

Define proteome

A

All the proteins present in a cell at a particular time

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2
Q

Define metabolome

A

The total number of metaboolites present in a cell (or tissue or organism)at a particular time

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3
Q

What are the transcriptome and the proteome linked by?

A

Codons in mRNA

  • Generally considered standard
  • Some exceptions e.g. mitochondrail genomes deviate slightly
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4
Q

What are the main features of the study of proteins?

A
  • Study of purified proteins: function/biochemistry in isolation, separation based on charge, size, antigenicity, tags
  • Study of their expression in vivo or in relation to disease
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5
Q

What is SDS-PAGE?

A

SDS - Polyacrylamide Gel Electrophoresis

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6
Q

What is SDS-PAGE used for?

A

One dimensional protein separation

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7
Q

How does SDS-PAGE work?

A
  • Based on charge:mass ratio
  • Applied electric field
  • Stacking gel to ensure all proteins start at same point
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8
Q

What is SDS?

A
  • A detergent used to keep all proteins soluble

- Denatures proteins

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9
Q

How are 2D-PAGE and SDS-PAGE used together?

A
  • 2D separates proteins ofsimilar masses by number of charges within protein along pH gradient with applied electrical charge
  • Then use SDS-PAGE to separate based on mass
  • The strip used in 2D is used like a stacking gel , placed on top and separated by charge
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10
Q

What is the benefit of using SDS and 2D PAGE together?

A

Allows more separation of proteins that would usually overlap
- Allows identification of partial peptide sequences from protein samples

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11
Q

How can proteomes be compared?

A
  • Compare matched gels for different samples
  • Label proteins and run on same separation
  • Differences in form/mass spec
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12
Q

What is a 2D separation modem used for?

A
  • To go direct from protein mixture to identification

- Could be used in rapid diagnostics

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13
Q

What does the 2D separation modem rely on?

A

Knowing the sequences of proteins

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14
Q

How does the 2D separation modem work?

A
  • IEF separation by HPLC in capillary tube
  • Then protein determination by mass spec, matrix assisted time of flight (MALDI-TOF) or by ES electro-spray technology
  • Utilises high performance liquid chromatography
  • Range of solutions, column with solid matrix
  • Start with 100% solution A, mix until 100% solution B
  • As pH changes, proteins come off based on charge
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15
Q

How can direct comparisono of proteomes by 2D-PAGE be done?

A
  • Overlay images

- Label proteins with colours, run on same gel

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16
Q

What are the limitations of SDS-PAGE?

A
  • Based on charge:mass ratio

- Many proteins overlap in charge and size so poor resolution

17
Q

What are the limitations of 2D-PAGE?

A
  • Not all proteins will run in isoelectric focussing
  • Amount of material limited
  • Need to harvest organelles to measure proteomes
  • Solutions to “stop cellular processes”
  • Proteases may degrade protein of interest before it reaches the lab
  • Large amounts of data
  • No information on biochem
18
Q

Give examples of protein analysis techniques

A
  • Refractometry
  • ELISA
  • Protein array
19
Q

What are the limitations of refractometry

A
  • Affected by what else is in sample

- Does not say what is there but what kind of shift has occured (more or less “stuff” than normal)

20
Q

What are the limitations of ELISA?

A
  • Shows presence not amount

- Requires knowledge of what protein and correct antibody

21
Q

What is the function of Western Blots?

A

Used to detect specific proteins by antibody

22
Q

Describe the process of a Western Blot

A
  • Transfer proteins from gel to filter
  • Block filter to avoid non-specific binding
  • Probe with antibody to protein of interest added
  • Proteomics used to remove protein from cell, antibody to detect protein,then separate on normal gel
23
Q

Give examples of uses of proteomics in clinical scenarios

A
  • Western blots
  • Snap tests for specific antigens and antibodies
  • To gain more information about disease processes e.g. protein profiling of urine from dogs with renal disease
  • Identification of cancer
  • IDentificatino of causes/diagnosis
  • Virulence/resistance mechanisms (requires hypotheses on what can be done, targets for control)
24
Q

What are some uses of metabolomics in clinical scenarios?

A
  • To establish why unpredictable effects may have occured

- To reformulate drug to avoid negative effects

25
Q

How can DNA sequence beused to gain information about proteins?

A
  • Genome = sequence, promotes and genes etc, converted to structural/messenger RNA
  • Can be used to model the RNA
  • RNA can be used to predict primary peptide sequences
  • Structural modelling can be done based on similar primary sequences
  • Structural prediciton based on primary peptide sequence
  • However presence of RNA does not mean a protein will be expressed from it, ot that the protein will be functional

URI (15 decks)

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