Omics in health and disease
Proteomics: Into part I
Proteomics: Large-scale study of proteins, particularly their structures and functions
This can be studied using:
- Yeast two-hybrid
- Protein microarrays
- Phage display
- Mass spectrometry
Applications of mass spectrometry-based proteomics include:
1. post-translational modifications
2. Protein-protein interactions
3. Organelle specific protein expression (e.g. mitochondria etc)
4. Effects of miRNAs on protein abundance
5. etc.
Proteomics research-aims are to:
- Identify large proportions of the proteome
- Identify protein modifications
- Quantify proteins
- Characterize protein interactions
Previously, 2D-gels were used to study proteomics
Advantage: Different forms of the same protein appear in different spots
But, there are also limitations
- Poor reproducibility
- Poor dynamic range
- Poor coverage (max. 1000 protein identifications)
- Difficult quantification of proteins
- Problems to resolve highly acidic/basic proteins, or proteins with extreme size of
hydrophobicity
- Difficulties in automation → very low-throughput
Result: 2D-gels are hardly used anymore
Omics get more and more
complex if going down the
pyramid
Omics get more easier to study
going up the pyramid
So, the more complex omics are harder to study
In addition, the human proteome is very complex
,→ It gets difficult to identify proteins in low abundance in a background of
proteins in high abundance
DNA-sequencing (genomics) and mass spectrometry (proteomics) are highly
complementary
→ Integrate genomics + proteomics to get full overview
However, the transcriptome does not correlate well with the proteome
General workflow for bottom-up/shotgun MS-based proteomics
Important to now every step
1. Cell lysis
2. Protein digestion
it is important to choose a protease based on the experimental question
→ Most commonly used protease for mass spectrometry based proteomics
experiments = trypsin
- Digestion after every arginine/ lysine residue
- Average length of tryptic peptides = 10 aa residues
- There is a minimum of 6 aa needed to be able to identify an unique protein sequence
3. nano reversed-phase liquid chromatography (RP-HPLC)
To separate peptides, to not ‘shoot’ all peptides at once at the MS
- Peptides bind to a C18 matrix in hydrophilic acidic solvent (e.g. 0.5% acetic acid)
- When using an increasing concentration of organic solvents (often hydrophilic),
peptides are separated according to their hydrophobicity.
- Hydrophilic peptides will elute first
, - Hydrophobic peptides have a stronger interaction with the matrix and elude
later
→ this ensures separation over time
Why nano? when using a thin capillary; smaller means lower flow rate, and thus a higher
intensity and faster eluding process.
4. Electrospray ionization (ESI)
The liquid droplets from the RP-HPLC turn into gas phase: this is called the electrospray
- When the liquid droplets evaporate during the gas phase, this results in peptides
containing protons: thus + charged
- the MS contains a pump with - charge, attracting the + charged peptides
This is a ionization source
5. Mass-spectrometer
There are 3 basic components in a mass spectrometer:
- An ionization source
- A mass analyzer
- And a detector
→ Mass analyzers measure the mass-to-charge (m/z) ratios of gas phase ions in
order to seperate them from each other
- each analyzer has it ons strengths and weaknesses, most mass spectrometers use
two or more mass analyzers
The importance of high resolution
→ Resolution or resolving power (RP):
the accuracy of measuring peptide
- Can be calculated by: (RP)=m/dm
Why is resolution important?
→ 2 peptides with similar mass
- increasing the resolution results in two
distinct peaks for each peptide
However, scanning at a higher resolution
comes at a cost: time!
There are different types of mass analyzers
1. TOF: Time of Flight
- measuring time it takes for a peptide to go through a tube; which correlates
with mass
2. Quadrupoles and longtraps
3. Orbitrap
- Ions are electrostatically trapped in an orbit around a central, spindle shaped
electrode (--> no magned needed)
- An image current is induced by passing by ions at the detector plates
- m/z is determined by fourier transformation of the periodic signal
- This gives a high sensitivity and a high resolution
→ The orbitrap is the most dominant mass analyzer in modern
proteomics
, (orbitrap principle in short: + charged ions osculate around central electrode
with different frequencies: this correlates with mass and can be translated into
m/z)
Hybrid orbitrap set-ups at the RIMLS
→ Orbitrap + Quadrupole
Quadrupole:
- Is used to isolate 1 specific peptide species
- → then fragmentation (in collision cell)
- you do this to identify aa sequence
The MS can switch between mass analysers between seconds
6. Full MS scan (Orbitrap)
If you obtain a full scan at the end of your MS-run, you will not see 1 single peak per peptide
but an isotope cluster.
- Masses are recorder with an accuracy below 1 parts per million (pmm)
- A peptide of 1000 Dalton is therefore measured at 1000 +/- 0.001 Dalton
You can also obtain a fragmentation spectrum (MS/MS) of a single peptide, this is used to
identify the specific aa sequence
isotope cluster
High resolution MS distinguishes between isotopes,but what are these different isotope
peaks you see?
→ they are due to carbon
normally: 12C
But there is a 1% change of 13C, so by change, 1 of the peptides will carry a 13C
Peptide charge following Electrospray ionization (ESI)
→ tryptic peptides are primarily double charged (but other charge states are
present)
- N terminus contains NH3+
- C terminus contains NH3+
But a H can also carry extra protons, so peptides can be seen in different charge states
Also, higher charges (3+ or higher) are also possible when:
- there is a missed cleavage → internal R/K
- PTMs
- Histidine side-chain
The measured value in a mass spectrometer is mass (m) divided by charge (z) of the
peptide (which is the number of attached protons) = m/z
Isotope cluster
→ Between peaks: mass difference is always 1 Da, so m=1
Then you look at the difference between the first two peaks: if this difference is 0.33
then m/z=0.33 → 1/z=0.33 → so z=3
If you then want to determine the mass, you multiply the monoisotopic peaks by the charge
(in this case 3), and then subtract the amount of protons (3)
Proteomics: Into part I
Proteomics: Large-scale study of proteins, particularly their structures and functions
This can be studied using:
- Yeast two-hybrid
- Protein microarrays
- Phage display
- Mass spectrometry
Applications of mass spectrometry-based proteomics include:
1. post-translational modifications
2. Protein-protein interactions
3. Organelle specific protein expression (e.g. mitochondria etc)
4. Effects of miRNAs on protein abundance
5. etc.
Proteomics research-aims are to:
- Identify large proportions of the proteome
- Identify protein modifications
- Quantify proteins
- Characterize protein interactions
Previously, 2D-gels were used to study proteomics
Advantage: Different forms of the same protein appear in different spots
But, there are also limitations
- Poor reproducibility
- Poor dynamic range
- Poor coverage (max. 1000 protein identifications)
- Difficult quantification of proteins
- Problems to resolve highly acidic/basic proteins, or proteins with extreme size of
hydrophobicity
- Difficulties in automation → very low-throughput
Result: 2D-gels are hardly used anymore
Omics get more and more
complex if going down the
pyramid
Omics get more easier to study
going up the pyramid
So, the more complex omics are harder to study
In addition, the human proteome is very complex
,→ It gets difficult to identify proteins in low abundance in a background of
proteins in high abundance
DNA-sequencing (genomics) and mass spectrometry (proteomics) are highly
complementary
→ Integrate genomics + proteomics to get full overview
However, the transcriptome does not correlate well with the proteome
General workflow for bottom-up/shotgun MS-based proteomics
Important to now every step
1. Cell lysis
2. Protein digestion
it is important to choose a protease based on the experimental question
→ Most commonly used protease for mass spectrometry based proteomics
experiments = trypsin
- Digestion after every arginine/ lysine residue
- Average length of tryptic peptides = 10 aa residues
- There is a minimum of 6 aa needed to be able to identify an unique protein sequence
3. nano reversed-phase liquid chromatography (RP-HPLC)
To separate peptides, to not ‘shoot’ all peptides at once at the MS
- Peptides bind to a C18 matrix in hydrophilic acidic solvent (e.g. 0.5% acetic acid)
- When using an increasing concentration of organic solvents (often hydrophilic),
peptides are separated according to their hydrophobicity.
- Hydrophilic peptides will elute first
, - Hydrophobic peptides have a stronger interaction with the matrix and elude
later
→ this ensures separation over time
Why nano? when using a thin capillary; smaller means lower flow rate, and thus a higher
intensity and faster eluding process.
4. Electrospray ionization (ESI)
The liquid droplets from the RP-HPLC turn into gas phase: this is called the electrospray
- When the liquid droplets evaporate during the gas phase, this results in peptides
containing protons: thus + charged
- the MS contains a pump with - charge, attracting the + charged peptides
This is a ionization source
5. Mass-spectrometer
There are 3 basic components in a mass spectrometer:
- An ionization source
- A mass analyzer
- And a detector
→ Mass analyzers measure the mass-to-charge (m/z) ratios of gas phase ions in
order to seperate them from each other
- each analyzer has it ons strengths and weaknesses, most mass spectrometers use
two or more mass analyzers
The importance of high resolution
→ Resolution or resolving power (RP):
the accuracy of measuring peptide
- Can be calculated by: (RP)=m/dm
Why is resolution important?
→ 2 peptides with similar mass
- increasing the resolution results in two
distinct peaks for each peptide
However, scanning at a higher resolution
comes at a cost: time!
There are different types of mass analyzers
1. TOF: Time of Flight
- measuring time it takes for a peptide to go through a tube; which correlates
with mass
2. Quadrupoles and longtraps
3. Orbitrap
- Ions are electrostatically trapped in an orbit around a central, spindle shaped
electrode (--> no magned needed)
- An image current is induced by passing by ions at the detector plates
- m/z is determined by fourier transformation of the periodic signal
- This gives a high sensitivity and a high resolution
→ The orbitrap is the most dominant mass analyzer in modern
proteomics
, (orbitrap principle in short: + charged ions osculate around central electrode
with different frequencies: this correlates with mass and can be translated into
m/z)
Hybrid orbitrap set-ups at the RIMLS
→ Orbitrap + Quadrupole
Quadrupole:
- Is used to isolate 1 specific peptide species
- → then fragmentation (in collision cell)
- you do this to identify aa sequence
The MS can switch between mass analysers between seconds
6. Full MS scan (Orbitrap)
If you obtain a full scan at the end of your MS-run, you will not see 1 single peak per peptide
but an isotope cluster.
- Masses are recorder with an accuracy below 1 parts per million (pmm)
- A peptide of 1000 Dalton is therefore measured at 1000 +/- 0.001 Dalton
You can also obtain a fragmentation spectrum (MS/MS) of a single peptide, this is used to
identify the specific aa sequence
isotope cluster
High resolution MS distinguishes between isotopes,but what are these different isotope
peaks you see?
→ they are due to carbon
normally: 12C
But there is a 1% change of 13C, so by change, 1 of the peptides will carry a 13C
Peptide charge following Electrospray ionization (ESI)
→ tryptic peptides are primarily double charged (but other charge states are
present)
- N terminus contains NH3+
- C terminus contains NH3+
But a H can also carry extra protons, so peptides can be seen in different charge states
Also, higher charges (3+ or higher) are also possible when:
- there is a missed cleavage → internal R/K
- PTMs
- Histidine side-chain
The measured value in a mass spectrometer is mass (m) divided by charge (z) of the
peptide (which is the number of attached protons) = m/z
Isotope cluster
→ Between peaks: mass difference is always 1 Da, so m=1
Then you look at the difference between the first two peaks: if this difference is 0.33
then m/z=0.33 → 1/z=0.33 → so z=3
If you then want to determine the mass, you multiply the monoisotopic peaks by the charge
(in this case 3), and then subtract the amount of protons (3)
