Les 2: 18/12
Mass spectrometry in proteomics - Varia1ons on MS
Adapted mass spectrometers for the study of protein complex
Mass spectrometers for measurement of large proteins
- ESI: very so- ioniza2on method -> na2ve interac2ons between molecules can o-en be
preserved.
o If you have to proteins that bind to each other, strong to each other -> preserved
during the ioniza2on.
- Mul2ple charges that reside on the complexes -> m/z value will drop -> come in
working window of the mass spectrometry
- the range of the mass analyzer.
o Mul2ple charges on the complex -> m/z value will drop. It will come in the working
flow of the mass analyzer.
- ESI normally occurs with a heated gas or a-er addi2on of an organic acid -> denatured
molecules. However, nanoflow HPLC -> evapora2on without heated gas or organic
solvent since droplets are much smaller.
o Denatura2on and dissocia2on of the two proteins
o Nanoflow HPLC = very small columns with very small diameters -> no need for
heated gas and acidic compound; droplets are so small that the analytes become
available without heated gas and acidic compound
o You can even measure the tetramer, only if it has enough charges
- We have seen already MS and MS-MS, but you can do many more with mass
spectrometry.
- Not trypsinizing the proteins -> introduce a complete protein in the MS or even a
protein complex -> CD collision -> ab dissociates of the an2gen => work with proteins.
It is possible to measure complete proteins for iden2fica2on, for protein complex
analysis, binding studies. You have to adapt the MS a liRle bit
- In this case, very large ions have to pass the quadrupole that is made for selec2on of
ions in the mass range 500-5000. Moreover, protein complexes seemed to have less
charges than expected, resul2ng in large m/z values. Quadrupoles with adapted
radiofrequencies (about 3-fold reduc2on), allowing m/z values 2ll 32.000.
o Then we have a larger working range -> you can look at larger protein complexes
- Dissocia2on of complex ions in a CID occurs, analogous to pep2de dissocia2on, by
collision with an inert gas. Resul2ng products are highly charged monomers that were
split off, and accordingly weakly charged ‘stripped’ oligomers. The sum of the charges
is the original charge on the precursor ion
o Nanoflow ioniza2on flow. You have spontaneous disolva2on and then the ion
enters the quadrupole that is set in such a way that allows for a higher m/z. Then
the proteins complex transvers to the collision cell and the complex is dissociated
-> no covalent bound broken, but electrosta2c bounds
o Your subunit proteins enter the TOF where they can be detected
- Be aware!
o Contribu2on of the different bonds to the protein-protein interac2on can vary
when complex is in the gas phase. Charge-charge and hydrophobic interac2ons
usually increase and decrease, resp..
, o Complex evaporated and ionized in a gas phase: the contribu2on of the bounds
differs -> molecules not surrounded by water anymore
o Contribu2on of the hydrogen bound decreases while the contribu2on of the
charge-charge interac2ons increases
- Example:
o MS/MS spectrum of:
§ a) half a proteasome (mutant, a7:b7),
§ c) free proteasome (a7:b7:b7:a7) and
§ d) a proteasome, bound to 4 cytochrome-c molecules.
- A-er CID:
o Removal of subunits on the exterior of the complex:
§ β-subunits are preferen2ally removed from the halved proteasome complex
(a)
• one subunit is should off. It has 20+ charge and the rest has 30+ charge ->
the number of charges is maintained during the process
§ α-subunits from the complete complex (C)
• the a-b connec2on is weaker compared to the b-b connec2on
o Removing the α-subunits allows for removing the inside substrate (cyt-c) (d)
§ Only when the alfa subunit is removed the cytochrome could removed.
- Note that the removed monomer is highly charged (shi- to the le-), while the
“stripped” oligomer is weakly charged (shi- to the right)
- Applica2ons:
o Stoichiometry and heterogeneity of protein complexes
o Dynamic interac2ons within complexes (log-on and –off of proteins, substrate
binding …)
§ By increasing the energy by CDI you can see more and more an2gen shouted
off by the an2body and then you can determine the affinity of the an2body
to the an2gen
Ion-mobility mass spectrometry (IMS)
Principle
- No mass spectrometry sensu strictu, but can easily be coupled to a mass spectrometer
(IMMS)
- Separa2on of ions is based on interac2on with a buffer gas, hence separa2on on the
basis of MW and shape. This means that isobaric molecules can be separated = one
more dimension!
o Isobaric molecules, molecules with exactly the same molecular weight, can be
separated in a MSI -> big advantage
o One kind of separa2on more, we have a dimension more
- Proteins or pep2des that are more compact show a higher mobility, allowing for the
separa2on of molecules with different conforma2ons.
- Same molecular weight but have another shape. When this molecule, the stripe,
travels through to a gas it will be slower than a circular molecule
Mass spectrometry in proteomics - Varia1ons on MS
Adapted mass spectrometers for the study of protein complex
Mass spectrometers for measurement of large proteins
- ESI: very so- ioniza2on method -> na2ve interac2ons between molecules can o-en be
preserved.
o If you have to proteins that bind to each other, strong to each other -> preserved
during the ioniza2on.
- Mul2ple charges that reside on the complexes -> m/z value will drop -> come in
working window of the mass spectrometry
- the range of the mass analyzer.
o Mul2ple charges on the complex -> m/z value will drop. It will come in the working
flow of the mass analyzer.
- ESI normally occurs with a heated gas or a-er addi2on of an organic acid -> denatured
molecules. However, nanoflow HPLC -> evapora2on without heated gas or organic
solvent since droplets are much smaller.
o Denatura2on and dissocia2on of the two proteins
o Nanoflow HPLC = very small columns with very small diameters -> no need for
heated gas and acidic compound; droplets are so small that the analytes become
available without heated gas and acidic compound
o You can even measure the tetramer, only if it has enough charges
- We have seen already MS and MS-MS, but you can do many more with mass
spectrometry.
- Not trypsinizing the proteins -> introduce a complete protein in the MS or even a
protein complex -> CD collision -> ab dissociates of the an2gen => work with proteins.
It is possible to measure complete proteins for iden2fica2on, for protein complex
analysis, binding studies. You have to adapt the MS a liRle bit
- In this case, very large ions have to pass the quadrupole that is made for selec2on of
ions in the mass range 500-5000. Moreover, protein complexes seemed to have less
charges than expected, resul2ng in large m/z values. Quadrupoles with adapted
radiofrequencies (about 3-fold reduc2on), allowing m/z values 2ll 32.000.
o Then we have a larger working range -> you can look at larger protein complexes
- Dissocia2on of complex ions in a CID occurs, analogous to pep2de dissocia2on, by
collision with an inert gas. Resul2ng products are highly charged monomers that were
split off, and accordingly weakly charged ‘stripped’ oligomers. The sum of the charges
is the original charge on the precursor ion
o Nanoflow ioniza2on flow. You have spontaneous disolva2on and then the ion
enters the quadrupole that is set in such a way that allows for a higher m/z. Then
the proteins complex transvers to the collision cell and the complex is dissociated
-> no covalent bound broken, but electrosta2c bounds
o Your subunit proteins enter the TOF where they can be detected
- Be aware!
o Contribu2on of the different bonds to the protein-protein interac2on can vary
when complex is in the gas phase. Charge-charge and hydrophobic interac2ons
usually increase and decrease, resp..
, o Complex evaporated and ionized in a gas phase: the contribu2on of the bounds
differs -> molecules not surrounded by water anymore
o Contribu2on of the hydrogen bound decreases while the contribu2on of the
charge-charge interac2ons increases
- Example:
o MS/MS spectrum of:
§ a) half a proteasome (mutant, a7:b7),
§ c) free proteasome (a7:b7:b7:a7) and
§ d) a proteasome, bound to 4 cytochrome-c molecules.
- A-er CID:
o Removal of subunits on the exterior of the complex:
§ β-subunits are preferen2ally removed from the halved proteasome complex
(a)
• one subunit is should off. It has 20+ charge and the rest has 30+ charge ->
the number of charges is maintained during the process
§ α-subunits from the complete complex (C)
• the a-b connec2on is weaker compared to the b-b connec2on
o Removing the α-subunits allows for removing the inside substrate (cyt-c) (d)
§ Only when the alfa subunit is removed the cytochrome could removed.
- Note that the removed monomer is highly charged (shi- to the le-), while the
“stripped” oligomer is weakly charged (shi- to the right)
- Applica2ons:
o Stoichiometry and heterogeneity of protein complexes
o Dynamic interac2ons within complexes (log-on and –off of proteins, substrate
binding …)
§ By increasing the energy by CDI you can see more and more an2gen shouted
off by the an2body and then you can determine the affinity of the an2body
to the an2gen
Ion-mobility mass spectrometry (IMS)
Principle
- No mass spectrometry sensu strictu, but can easily be coupled to a mass spectrometer
(IMMS)
- Separa2on of ions is based on interac2on with a buffer gas, hence separa2on on the
basis of MW and shape. This means that isobaric molecules can be separated = one
more dimension!
o Isobaric molecules, molecules with exactly the same molecular weight, can be
separated in a MSI -> big advantage
o One kind of separa2on more, we have a dimension more
- Proteins or pep2des that are more compact show a higher mobility, allowing for the
separa2on of molecules with different conforma2ons.
- Same molecular weight but have another shape. When this molecule, the stripe,
travels through to a gas it will be slower than a circular molecule
