L13 - High-throughput research: proteomics

Question 1

What is high throughput methodology?

Answer 1

Taking established methodology & re-applying it to an automated system

Question 2

Transcriptome

Answer 2

mRNA molecules of the cell

Question 3

Metabolome

Answer 3

The metabolic products of the cell, that is, all the metabolites

Question 4

Secretome

Answer 4

The secreted proteins of a cell

Question 5

Phosphome

Answer 5

Total phosphorylated proteins of a cell

Question 6

Kinome

Answer 6

All kinases of the cell

Question 7

Is the genome constant in an organism?

Answer 7

YES

But the transcriptome & then therefore, all the others following, changes due to what is required by the cell

Bioinformatics can be seen as the control & analysis of the information

Question 8

What is proteomics?

Answer 8

The study of the proteome

Question 9

What is the proteome?

Answer 9

The total cellular protein content of the cell

Set of PROTEins encoded by the genOME

Question 10

Stages in examining the proteome

Answer 10

SEPARATE OR FRACTIONATE
– Common approach is to carry out electrophoresis
– Also possible to use liquid chromatography

IDENTIFICATION
– The pI (isoelectric point) & MW matches on the databases
– Western blot is possible to carry out
– Sequencing

Question 11

Often compare proteomes of cells in 2 different ‘states’ or origins

Answer 11

1) Separate proteins wither by 2DE Gel Electrophoresis of MDLC (Multi-dimentional liquid chromatography)
2) Compare & identify differences
3) Identify proteins
4) Form hypothesis

Question 12

Why would you compare proteomes?

Answer 12

Cells in different states have different proteomes – eg. disease or stage of development

If we can understand this change we may understand what is happening to the cell

Can be compared against a ‘standard’ in a database

Question 13

What is peptide mass finger-printing?

Answer 13

Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification in which the unknown protein of interest is first cleaved into smaller peptides, whose absolute masses can be accurately measured with a mass spectrometer

Question 14

General methodology of protein fingerprinting

Answer 14

1) Collect control and ‘state’ protein samples
2) Separate proteins - 2DE/chromatography
3) Compare control and ‘state’ gels to determine spots that have changed
4) Pick spots (i.e. cut out from gel!) that have changed
5) Digest the protein in the gel plug
6) Separate peptides by mass spectrometry
7) Identify proteins from mass of peptides produced
8) Carry out mass spectrometry peptide sequencing

Question 15

Why is it harder to prepare samples of proteins than DNA samples?

Answer 15

DNA is pretty robust & easy to purify - takes a lot to destroy it

Proteins are fragile – short lived & easy to break down – constantly modified by post-translational modifications

Have to be a lot more careful

Have to try to maintain the tertiary structure

Question 16

2-dimentional gel electrophoresis (2DE)

Answer 16

‘Traditional’ proteomic approach

Using large PAGE gels:
• Initially Separates by iso-electric focusing (IEF) - based on iso-electric point (ACROSS)
• Second separation based on molecular weight (DOWN)

Question 17

Multi-dimensional liquid chromatography (MDLC)

Answer 17

Application of an ‘old’ technology to a ‘new’ problem

1) Protein sample separated by passing over a 1st column
2) These fractions are then separated by passing over a 2nd different column
3) Changes can be monitored by UV trace
4) Can be ‘automatically’ loaded in to mass-spec