Global analysis of functionally relevant proteins for biomarker and drug target
discovery
Proteomics = The large-scale analysis of proteins
- Expression
- Post-translational modifications
- Protein-protein interactions
If we look at how many different proteins there are, compared to RNA or DNA, proteins are
the majority.
DNA: 19.629
RNA: ~90.000 isoforms
Protein: 15.721 observed; 11.353 isoforms; 100.000 post-translational modifications
Mass spectrometry (MS)
Using this technique, scientists can get insight in the following tissues:
- Tumour tissue
- Urine
- Faeces
- Liquor
- Blood platelets
Eventually, this technique could contribute to the early detection of cancer or personalized
therapy. In the picture depicted under here, the blue circled is the process of mass
spectrometry in a nutshell.
→A mass spectrometer is a machine used to weigh molecules on
a molecular scale
→It measures peptide masses and peptide fragment masses
1
,Steps in proteomics experiment
First the proteins need to be isolated and be digested to small peptides. This way, they can
be fractionated by liquid chromatography.
Protein identification and quantification
The mass analyzer scans which peptides molecular weight are eluting from the column.
Later, via database research, the molecular weight information is matched to protein
sequences.
Data mining: from data to novel insights
To analyse data, there are multiple techniques:
In the picture right, the proteins that are overexpressed and under
expressed are not clustered.
This picture shows a hierarchical cluster: this example is from the
BRCA gene and its mutations (seen in breast cancer). The red clusters
mean those proteins are downregulated.
2
, Proteomics for biomarker discovery
Biomarker = a biomarker is a defined characteristic that is measured as an indicator of
normal biological processes, pathogenic processes, or a response to an exposure or
intervention.
• Most biomarkers are measured in blood
• Most biomarkers are proteins
➔ Example: if people are screened for familiar breast cancer, BRCA mutation is a
biomarker, because this mutation is known to be present in breast cancer patients.
Multiple applications for biomarker-based diagnostics of disease
The picture above shows different applications where biomarkers can be used. The more
you go to the right, this shows an application that costs a lot.
Plasma proteomics
This is challenging, because there is a wide dynamic range of protein concentrations.
Strategies for biomarker discovery in plasma:
- Protein fractionation
- Peptide fractionation
- Abundant protein depletion
- Enrichment of vesicles
Biomarker discovery by proteomics
This also consists of several steps;
For discovery, a clinical need sample source is necessary. This discovery can be done with a
mass spectrometer. Also validation is done with a MS. Assay development too, but (!) an
alternative is antibody based screening.
3
discovery
Proteomics = The large-scale analysis of proteins
- Expression
- Post-translational modifications
- Protein-protein interactions
If we look at how many different proteins there are, compared to RNA or DNA, proteins are
the majority.
DNA: 19.629
RNA: ~90.000 isoforms
Protein: 15.721 observed; 11.353 isoforms; 100.000 post-translational modifications
Mass spectrometry (MS)
Using this technique, scientists can get insight in the following tissues:
- Tumour tissue
- Urine
- Faeces
- Liquor
- Blood platelets
Eventually, this technique could contribute to the early detection of cancer or personalized
therapy. In the picture depicted under here, the blue circled is the process of mass
spectrometry in a nutshell.
→A mass spectrometer is a machine used to weigh molecules on
a molecular scale
→It measures peptide masses and peptide fragment masses
1
,Steps in proteomics experiment
First the proteins need to be isolated and be digested to small peptides. This way, they can
be fractionated by liquid chromatography.
Protein identification and quantification
The mass analyzer scans which peptides molecular weight are eluting from the column.
Later, via database research, the molecular weight information is matched to protein
sequences.
Data mining: from data to novel insights
To analyse data, there are multiple techniques:
In the picture right, the proteins that are overexpressed and under
expressed are not clustered.
This picture shows a hierarchical cluster: this example is from the
BRCA gene and its mutations (seen in breast cancer). The red clusters
mean those proteins are downregulated.
2
, Proteomics for biomarker discovery
Biomarker = a biomarker is a defined characteristic that is measured as an indicator of
normal biological processes, pathogenic processes, or a response to an exposure or
intervention.
• Most biomarkers are measured in blood
• Most biomarkers are proteins
➔ Example: if people are screened for familiar breast cancer, BRCA mutation is a
biomarker, because this mutation is known to be present in breast cancer patients.
Multiple applications for biomarker-based diagnostics of disease
The picture above shows different applications where biomarkers can be used. The more
you go to the right, this shows an application that costs a lot.
Plasma proteomics
This is challenging, because there is a wide dynamic range of protein concentrations.
Strategies for biomarker discovery in plasma:
- Protein fractionation
- Peptide fractionation
- Abundant protein depletion
- Enrichment of vesicles
Biomarker discovery by proteomics
This also consists of several steps;
For discovery, a clinical need sample source is necessary. This discovery can be done with a
mass spectrometer. Also validation is done with a MS. Assay development too, but (!) an
alternative is antibody based screening.
3
