SMV EXPERIMENTAL CELL BIOLOGY II
Neuroproteomics
Proteomics using mass spectrometer
With proteomics you quantify proteins in the tissue using a mass spectrometer. Analytes can
be proteins, peptides or more specific carbohydrates, fat, small molecules or chemicals.
You inject analytes in the mass spectrometer which reach a detector. There are two
parameters: mass of analytes and the intensity of the analytes (lower concentrations is lower
intensity). Proteins consist of amino acids who all have a unique mass. Most of the time
when identifying proteins we first cut them into peptides. This is because the mass
spectrometer can identify peptides with a high sensitivity, and proteins are measured with
low resolution and poor mass accuracy. The proteins are digested into peptides with Trypsin:
cleaves specifically the peptide bond between the carboxyl group of arginine, or the carboxyl
group of lysine, and the amino group of the adjacent amino acid. If you identify the peptides
you can identify the protein.
However, there is one problem. The mass spectrometer can not distinguish between two
peptides when they have the same mass. This is why we also need to identify the amino
acid sequence. This is done by a tandem mass spectrometer, two mass spectrometers
joined together. After the mass is detected, the peptides are filtered. One peptide goes into
the fragmentation chamber where it will absorb energy. This leads to the peptide breaking in
fragments where the energy is the weakest: the peptide bond. Now the peptide is broken
into fragments, you get N- and C-terminal fragments. Each amino acid has a mass, so by
subtracting the masses of fragment ions we can deduce the sequence of the peptide.
Normally you will have over 3000 proteins in a sample which generates many peptides. This
is very complex, so to reduce this you can couple MS to liquid chromatography. Most of the
time this is enough, but if the sample is still too complex you can use SDS-page: cut gel into
several slices and perform MS analysis separately to further reduce sample complexity.
With high pressure liquid chromatography you fractionate peptides to reduce complexity.
You pump liquid into the separation column where the peptides will seperate. This is based
on hydrophobicity of the amino acids of the peptides. Hydrophilic peptides will come out of
the column earlier than the hydrophobic ones.
Mass spectrometer: the hardware
The basics:
,In MS peptides have to be in the gas phase. Two different techniques to get them from
solvents to gas phase:
- MALDI
- Electrospray ionization (ESI): the effluent leaves ESI needles as charged droplets. In
repeated evaporation steps the solvent is evaporated and a charged molecule enters
the mass spectrometer.
Peptides don’t have any energy from themselves, but in the mass spectrometer a pusher
gives all the peptides the same energy. Because of the difference in mass, the velocity of the
peptides will also be different. Bigger peptides move slower and the other way around. If we
measure the time it takes to reach the detector we can identify the mass.
Why do we still study proteomics and not only transcriptomics? This is because
protein/mRNA correlation is extremely low. The protein concentration depends on different
factors. With proteomics you can also study post-translational modifications.
Proteomics dissection of Alzheimer’s disease
Alzheimer’s disease (AD) has the most common symptoms of dementia: memory
impairment, disorientation, personality changes and cognitive decline. Definitive diagnosis of
AD is only possible post-mortem and there is no cure available. The etiology is largely
unknown, you can have mutations in APP, PSEN1 and PSEN2. The major risk factors are
age and APOE4. The pathological hallmarks are Tau tangles and Aβ plaques.
There are two types of AD: familial and sporadic. Familial has a young onset of 50-60 year
and you can distinguish by mutations in APP/PSEN1/2. In most cases AD is sporadic and
has an old onset.
The most famous hypothesis for the cause of AD is the amyloid cascade hypothesis. This is
that aggregation of Aβ causes cellular changes which leads to synapse loss/neuron death
which causes cognitive impairment. Then the cure for AD would be to get rid of the Aβ
oligomet. However, it is not that simple, AD is a multifactorial disease.
Proteomics was used to reveal AD pathology. See which proteins are involved in Aβ
plaques, Aβ accumulation in brain vasculature, neurofibrillary tangles and granulovacuolar
degeneration.
AD pathology is staged in Braak stages and highly correlates with the clinical symptoms.
Proteomics of the Braak stages can be crucial in detecting AD before neuron death so it can
be cured.
, Dynamic leukocyte-endothelial cell interactions
Capillaries are where everything takes place, blood cells leave the circulation and go into the
tissue. Not the whole vessel wall is open, they leave at certain sites. The blood cells can go
upstream or downstream of the vessel walls.
Leukocyte transendothelial migration happens with:
- Immune surveillance
- Inflammation
- Cancer metastasis
- Immune diseases (rheumatoid arthritis)
- Stem cell homing: find their way back to the bone marrow after stem cell
transplantation.
- Atherosclerosis
The first step of the process of transendothelial migration is when neutrophils start rolling
over the endothelial layer of the blood vessel. When the endothelial layer is inflamed, they
express molecules where neutrophils can bind to. Neutrophils can then cross the layer,
through 1 (transcellular) or in between 2 cells (paracellular). In vitro you can study this with
the vein of the umbilical cord.
Every different step of the migration is regulated by specific pools of proteins. The
endothelial layer is not flat when inflamed, small spikes/structures stick out. Neutrophils can
recognize them and bind to them. If more structures are present, more neutrophils cross
there (hotspot). Most neutrophils/monocytes migrate paracellular and T-cells mostly
transcellular, unknown why. It is a tight gap that neutrophils are going through. There is
maximum pore size in the endothelial which is the same for all the leukocytes. The pore
makes sure only leukocyte crosses and fluids won't leak. An actin ring keeps the gap at the
minimal size, actin dynamics are essential for transendothelial migration.
Actin in transendothelial migration
Actin is highly conserved and about 40% of the proteins in a cell. Actin can hydrolyze and
form a fiber/filament (f-actin). Can grow at plus end and hydrolyze at the minus end. A single
actin falls off at minus end after hydrolyzing ATP and can then bind to the plus side, so it
migrates. This pushes the membrane forward and causes membrane ruffling. This is
involved in a lot of cellular processes, such as vesicle forming and mitosis.
Actin polymerization is organized by small GTPases (RhoA, Rac1 and Cdc42). RhoA forms
actin stress fibers, Rac1 lamellipodia ad Cdc42 filopodia. When GDP is bound to a GTPase
Neuroproteomics
Proteomics using mass spectrometer
With proteomics you quantify proteins in the tissue using a mass spectrometer. Analytes can
be proteins, peptides or more specific carbohydrates, fat, small molecules or chemicals.
You inject analytes in the mass spectrometer which reach a detector. There are two
parameters: mass of analytes and the intensity of the analytes (lower concentrations is lower
intensity). Proteins consist of amino acids who all have a unique mass. Most of the time
when identifying proteins we first cut them into peptides. This is because the mass
spectrometer can identify peptides with a high sensitivity, and proteins are measured with
low resolution and poor mass accuracy. The proteins are digested into peptides with Trypsin:
cleaves specifically the peptide bond between the carboxyl group of arginine, or the carboxyl
group of lysine, and the amino group of the adjacent amino acid. If you identify the peptides
you can identify the protein.
However, there is one problem. The mass spectrometer can not distinguish between two
peptides when they have the same mass. This is why we also need to identify the amino
acid sequence. This is done by a tandem mass spectrometer, two mass spectrometers
joined together. After the mass is detected, the peptides are filtered. One peptide goes into
the fragmentation chamber where it will absorb energy. This leads to the peptide breaking in
fragments where the energy is the weakest: the peptide bond. Now the peptide is broken
into fragments, you get N- and C-terminal fragments. Each amino acid has a mass, so by
subtracting the masses of fragment ions we can deduce the sequence of the peptide.
Normally you will have over 3000 proteins in a sample which generates many peptides. This
is very complex, so to reduce this you can couple MS to liquid chromatography. Most of the
time this is enough, but if the sample is still too complex you can use SDS-page: cut gel into
several slices and perform MS analysis separately to further reduce sample complexity.
With high pressure liquid chromatography you fractionate peptides to reduce complexity.
You pump liquid into the separation column where the peptides will seperate. This is based
on hydrophobicity of the amino acids of the peptides. Hydrophilic peptides will come out of
the column earlier than the hydrophobic ones.
Mass spectrometer: the hardware
The basics:
,In MS peptides have to be in the gas phase. Two different techniques to get them from
solvents to gas phase:
- MALDI
- Electrospray ionization (ESI): the effluent leaves ESI needles as charged droplets. In
repeated evaporation steps the solvent is evaporated and a charged molecule enters
the mass spectrometer.
Peptides don’t have any energy from themselves, but in the mass spectrometer a pusher
gives all the peptides the same energy. Because of the difference in mass, the velocity of the
peptides will also be different. Bigger peptides move slower and the other way around. If we
measure the time it takes to reach the detector we can identify the mass.
Why do we still study proteomics and not only transcriptomics? This is because
protein/mRNA correlation is extremely low. The protein concentration depends on different
factors. With proteomics you can also study post-translational modifications.
Proteomics dissection of Alzheimer’s disease
Alzheimer’s disease (AD) has the most common symptoms of dementia: memory
impairment, disorientation, personality changes and cognitive decline. Definitive diagnosis of
AD is only possible post-mortem and there is no cure available. The etiology is largely
unknown, you can have mutations in APP, PSEN1 and PSEN2. The major risk factors are
age and APOE4. The pathological hallmarks are Tau tangles and Aβ plaques.
There are two types of AD: familial and sporadic. Familial has a young onset of 50-60 year
and you can distinguish by mutations in APP/PSEN1/2. In most cases AD is sporadic and
has an old onset.
The most famous hypothesis for the cause of AD is the amyloid cascade hypothesis. This is
that aggregation of Aβ causes cellular changes which leads to synapse loss/neuron death
which causes cognitive impairment. Then the cure for AD would be to get rid of the Aβ
oligomet. However, it is not that simple, AD is a multifactorial disease.
Proteomics was used to reveal AD pathology. See which proteins are involved in Aβ
plaques, Aβ accumulation in brain vasculature, neurofibrillary tangles and granulovacuolar
degeneration.
AD pathology is staged in Braak stages and highly correlates with the clinical symptoms.
Proteomics of the Braak stages can be crucial in detecting AD before neuron death so it can
be cured.
, Dynamic leukocyte-endothelial cell interactions
Capillaries are where everything takes place, blood cells leave the circulation and go into the
tissue. Not the whole vessel wall is open, they leave at certain sites. The blood cells can go
upstream or downstream of the vessel walls.
Leukocyte transendothelial migration happens with:
- Immune surveillance
- Inflammation
- Cancer metastasis
- Immune diseases (rheumatoid arthritis)
- Stem cell homing: find their way back to the bone marrow after stem cell
transplantation.
- Atherosclerosis
The first step of the process of transendothelial migration is when neutrophils start rolling
over the endothelial layer of the blood vessel. When the endothelial layer is inflamed, they
express molecules where neutrophils can bind to. Neutrophils can then cross the layer,
through 1 (transcellular) or in between 2 cells (paracellular). In vitro you can study this with
the vein of the umbilical cord.
Every different step of the migration is regulated by specific pools of proteins. The
endothelial layer is not flat when inflamed, small spikes/structures stick out. Neutrophils can
recognize them and bind to them. If more structures are present, more neutrophils cross
there (hotspot). Most neutrophils/monocytes migrate paracellular and T-cells mostly
transcellular, unknown why. It is a tight gap that neutrophils are going through. There is
maximum pore size in the endothelial which is the same for all the leukocytes. The pore
makes sure only leukocyte crosses and fluids won't leak. An actin ring keeps the gap at the
minimal size, actin dynamics are essential for transendothelial migration.
Actin in transendothelial migration
Actin is highly conserved and about 40% of the proteins in a cell. Actin can hydrolyze and
form a fiber/filament (f-actin). Can grow at plus end and hydrolyze at the minus end. A single
actin falls off at minus end after hydrolyzing ATP and can then bind to the plus side, so it
migrates. This pushes the membrane forward and causes membrane ruffling. This is
involved in a lot of cellular processes, such as vesicle forming and mitosis.
Actin polymerization is organized by small GTPases (RhoA, Rac1 and Cdc42). RhoA forms
actin stress fibers, Rac1 lamellipodia ad Cdc42 filopodia. When GDP is bound to a GTPase
