Why does quantitative Proteomics matter?
- Biological changes: Shows what goes up & down
- Mechanisms: shows pathway activation and regulation
- Clinical use needs accuracy, which MS quantitation provides
Describe absolute vs relative quantitation
Absolute: moles measured (protein concentration)
Relative: units measured (protein ratio, 2-fold more/les)
Describe the why you would use absolute or relative quantitation
Absolute:
-Mostly diagnostic –> industry&hospitals
- Is not always possible.
- Costs are higher and technically more challenging.
- Fewer proteins in single run.
Relative:
- Mostly discovery –> research labs
- Always possible.
- Lower cost but lower accuracy.
- Many protein in single run
Describe Label-based relative quantitation
Label-based methods use stable isotopes to distinguish peptides with the same mass from different
samples.
Peptides are labelled with identical reagents. Intensities can be compared if the signal corresponds to peptides with similar structure.
ITRAQ
Peptides with the same structure (m/z) but from different samples, are all labelled with this reporter group. In MS1 all peptides coelute, in MS2 see specific reporter groups & their abundance.
Done because adding all peptides together increases signal.
Reagent has 3 regions:
1. Mass reporter: reports when ITRAG reagent has been seen
2. Reactive terminus = sticks to peptide
3. Variable region: ensures for same mass of all reagents
Covalent labelling: TMTpro vs iTRAQ
TMTPRO can give higher plexing (more samples), better fragmentation and works across all analysis pipelines.
Describe Isotope labelleling
Used for absolute quantitation. In LC: elute at the exact same time, but in mz see the difference between ‘light’ and ‘heavy’
Light: C12+C13
Heavy: C13 added.
Gives quantitative (compare light to heavy) and qualitative (m/z, framentation etc) information
Describe Label-free relative quantitation
Each sample is analysed independently –> intensities normalised & compared –> large cohorts.
Label-free methods offer flexibility and scalability by analysing samples independently
and comparing them computationally.
- MS1 intensity & alignment (feature of healthy vs control)
- Spectral counting: Total nr of peptide-spectrum matches.
Describe how you can use TMTpro to measure 45 samples
- Create 1 Reference sample for all the 45 samples –> same volume of each sample to create ‘average’
- Make 3 runs all with TMTpro + Reference:
- Sample 1-15
- Sample 16-30
- Sample 31-45
In what proteins are we interested?
The ones that change in relative abundance over time, which is a very small amount
Describe AQUA and SISCAPA
AQUA: add ‘heavy’ identical peptide to endogenous peptide, measure ratio. Accounts for matrix effects etc. Limitation = cost, stability over time
SISCAPA: Add digestion of sample by antibody first, to enrich target peptides –> remove background. Then AQUA. Limitation = cost & time
What techniques are dominant for discovery proteomics (relative)?
DIA and TMTpro.
