Proteomics Kate Heesom L15

Question 1

What is the genome?

Answer 1

Provides an overall description of the complete set of genetic instructions that make up an organism

Question 2

How many coding genes need to make a tiny microscopic worm vs a human?

Answer 2

Twice as many coding genes for worm and less than twice the coding genes for a human.
Evolutionary and physiological complexity isn’t just a consequence of gene number. The increase in complexity we see as organisms evolve isn’t occurring solely at the level of the genome

Question 3

Functional Genomes?

Answer 3

Transcriptomics, proteomics, and metabolomics

Question 4

Difference between proteome and proteomics?

Answer 4

Proteome-the complete set of proteins that can be expressed from a genome
Proteomics-The study of the genome (Often used fir identifying proteins of interest such as drug targets, markers of disease onset/progression, research targets)

Question 5

What is the general proteomics strategy?

Answer 5

COMPLEX protein mixture

1) Reduce complexity/Separate proteins
2) Generate Peptides
3) Analyse by MS
4) Database mining for protein identification

Question 6

How might you reduce complexity of a protein mixture?

Answer 6

Sub cellular fractionation eg. purify poi from nuclei

Affinity approaches eg. pulling out phospho proteins in a cell

Question 7

1) Reducing complexity:

Answer 7

Polyacrylamide gel electrophoresis (SDS PAGE)
1D gel - separates proteins by MW but some proteins may be at the same MW
Can obtain complexity from amount of proteins visualised on the gel
Info on distribution of proteins of the MW within that sample
Quick and cheap
Only useful if sample is v.simple - separating single proteins

Question 8

How may you identify an in vitro drug target that is interacting with many proteins and causing side effects?
Useful as knowing the biology of the situation you are trying to treat and the structure of the drug so that you can manipulate the drug to only keep the beneficial interaction and can then remove the interactions causing side effects

Answer 8

1) Use solid support with drug attached in a column
2) Add in cell lysate containing all proteins through the column (BINDING)
3) Add in excess free drug that will compete with the binding reaction and drug receptor proteins will be released into solution and can then separate on the 1D gel (ELUTION)
4) 1D gel (couple of major bands-likely to be one isolated protein)
5) Analyse by mass spec
6) Database mining and identification

Only proteins that interact with the drug will bind to the column therefore only your drug receptor proteins stick onto the column and therefore all other proteins should run through the column (WASH). Using highly specific affinity approach we can pull out a very small subset of proteins

Question 9

2) Generating Peptides-what enzyme?

Answer 9

Use TRYPSIN (serine protease) which cuts at the C terminus of every lysine and arginine. Generates a very specific cleavage pattern.
Identifying proteins at the peptide level:
Add up the mass of these peptide fragments to get mass of protein (from MS)
Peptide mass fingerprint is unique to this protein (dependent on the seq of protein where K and R are present)

Question 10

3) What type of MS is used?
How does it work?
Do you use Crystals? YES

Answer 10

MALDI (Matrix assisted laser desorption ionisation) TOF (time of flight) MS
Use a MALDI target plate with a number of wells where you can add multiple different samples and analyse them in a single run
Well contains crystals of our peptides and matrix
Matrix is a chemical that absorbs energy at a wavelength characteristic of UV light from the laser.
Use a UV laser as an ionisation source on the plate
Peptides ionise and become charged.
They enter the TOF tube which is a mass analyser
Small proteins go quicker
Apply electrostatic protential between the target plate and aperture at from of the TOF tube
Detect using a detector

Question 11

What are the three key components to a MALDI TOF MS machine

Answer 11

1) ionisation source (UV laser)
2) time of flight tube (TOF)
3) mass detector

Question 12

What type of spectrometry should you use for a large sample of proteins?

Answer 12

Liquid chromatography MS-
Bind peptides to a column and eventually elute them by adding an increasing gradient of organic solvent. As you increase the organic solvent peptides elute and you get separation of peptides -flow them directly into an MS

Since you are eluting a large number of mixed peptides from multiple proteins you are unable to match this list of masses yet you hope to match them to a mass peptide fingerprint of one protein. Therefore you require an additional level of analysis MSMS spectra (isolate each peak shown in the MS1 spectra)

Question 13

MSMS

Answer 13

Spectra 1 (digest with trypsin) Spectra 2 = additional level of alanysis (collision induced dissociation - bombarding with He ions breaks up the individual peptide bonds between amino acids) A good MSMS spectra is for when we have broken at every potential peptide bond and you achieve a ladder effect of the different fragments (mass of peaks=mass of aa) gives us aa sequence of that peptide

Peptide sequence is now obtained you can identify which protein that peptide came from using a database

Question 14

What is the time frame for LC-MSMS

Answer 14

2hrs it is a constantly ongoing process

Question 15

LC-MSMS

Answer 15

thousands of peptide seqs generated during a single analysis to so potentially we can identify 1000s of proteins in that way.

Question 16

What are the application of LCMS?

Answer 16

Protein identification and protein quantification (useful for studying disease)

Question 17

What information is on a protein identification data report?

Answer 17

Coverage
List of identified proteins with their corresponding excision numbers which provide more info about their genome location etc
Coverage=% of the total aa seq of each protein found that was matched by peptides and actually identified and effectively sequenced during the analysis
normally high values ~70/80/90%
Can also be important when looking for subtle differences between proteins like distinguishing the presence of 2 diff isoforms in a sample

Question 18

How do u perform Protein identification using LCMS?

Answer 18

Global Proteome Profiling
Take a complex sample and identify every protein present within that sample

Run sample out onto 1D gel lane and cut gel into a number of slices rather than individual bands on the gel (into 10 equal pieces

Subject each piece of gel to an in-gel digestion with trypsin

LC-MS analysis

Can identify more than 1000 proteins from 1 slice
Recent expt identified >15,000 from a single sample

Question 19

How do u perform Protein quantification using LCMS?

Useful for studying disease

Answer 19

Two methods SILAC and TMT

Question 20

What is SILAC?

Answer 20

Stable isotope labelling with amino acids in culture
Most common quantitative proteomic approach
Comparing different conditions
e.g. using K0R0 light sample - condition1 and K6R6 - condition 2
Mix lysates
1D gel separates proteins (proteins from both conditions)
Cut gel lane into sections
In-gel digestion and Nano LC MSMS
6Da (differ in mass) between peaks on MS spectra-obtain pairs of peaks-pairs represent same peptide but from 2 different conditions

Can observe quantitation at MS1 level-further fragment peptides (MS2) to get seq and identify the protein the peptide has come from using a database

By looking at the relative intensities of the peaks in each pair we get a measure of the relative abundance of that peptide in different conditions

Question 21

What is shown in the SILAC data report?

Answer 21

Relative abundance between condition 1 and 2.

Question 22

What has been discovered using SILAC? David Matthews group

A more recent collaboration with Cambridge hopes to look at ebola proteins (non pathogenic and pathogenic form-why is this so deadly?)

Answer 22

Silac comparison of mock (light) and adenovirus (heavy) infected human cells.
Infection showed that in the heavy samples an increase in a ubiquitin ligase complex that specifically targets and degrades key human cellular components shown by a decrease.
These proteins may be targets for novel antiviral strategies

Question 23

What is TMT and how do you do it?

For protein quantification (A different approach to SILAC)

Answer 23

Tandem mass Tagging
Used for non-culterable samples (clinical samples e.g. tissues)
Samples are tagged POST extraction at the peptide level with 6 TMT tags
TMT tag: Reporter group:Cleavable linker:Mass Normaliser:Peptide Reactive group, - sticks to N terminus of every peptide
With TMT every peptide should be available for quantification. Each tag has a different reporter group.

1) Take samples to compare
2) Treat samples and isolate proteins (make a protein extract from each sample)
3) Denature, reduce, alkylate and trypic digest
4) Label peptides with 1 of the different TMT tags (1 tag per sample)
5) Quench the labelling rxn to avoid cross labelling
6) Combine the tagged samples together-contains all the peptides from all the samples we are comparing from
7) Analyse with LCMS
MS1-no info about sample these peptides have come from unlike the pairs in SILAC
8) Isolate peaks and fragment them (MSMS to break peptide bonds)
This has a dual purpose:
i)provides fragmentation MSMS spectra to get seq of peptide
ii) Results in cleavage of the TMT tag at linker region and releases the reporter groups

Question 24

In TMT why is the mass normaliser important?

Answer 24

Balances the overall mass of the tag between the different mass of the reporter groups so that the overall mass is constant. Therefore the total tag has a single mass regardless of which particular reporter group you have on it. Therefore only see one peak in MS1 (important as 6 separate peaks would over complicate it)
Overall mass of tag is constant therefore a given peptide will have exactly the same mass regardless from what sample it came from
Therefore in MS1 a given peptide will be given a single peak in the spectra

Question 25

What are the types of Tags in TMT?

Answer 25

126-132

Question 26

TMT MSMS

With TMT you get identification of 1000s of proteins in a sample with quantitative info

Answer 26

Obtain a seq of a particular peptide
Cleavage of linker region also releases the reporter groups - appear as a cluster at the end of the spectra

Like with our SILAC peaks where relative intensity = relative abundance
With TMT expts we also have the quantitative cluster which has the relative abundance of each peptide between the 6 conditions we are looking at.

Question 27

What do you see in a TMT data report?

Answer 27

Multiple different ratio columns and in the expt we have compared everything relative to the samples tagged with 126

You have a huge amount of information with this approach since you can compare multiple samples e.g. 6 different times or conditions

Question 28

What has Jan Fraynes group done using TMT?

Answer 28

Looking at whether we can make blood from stem cell sources rather than having to rely on donor blood
Comparison of proteomes of iPSCC derived erythroid cells and normal adult erythroid cells
Proteomes are v similar but more work is needed to elucidate the nature of the subtle changes before the iPSC derived cells can be considered for transfusion therapy

Question 29

Why use proteomics?

Answer 29

Transcript abundance does not equal protein abundance
In euk 1 gene gives rise to >1 proteins due to
Alternative Splicing
Alternative Transcriptional Start sites
Post translational modifications

Many changes in activity occur via protein modification (phosphorylation) or regulated localisation with no change in transcript abundance

Genomics and transcriptomics give no information about protein-protein interactions which are responsible for function.