Lecture 5: 8/11
Quiz
- List the different aspect of problems where proteomics can give answers to instead of
genomic or transcriptomics
o Alterna9ve splicing
o Protein iden9fica9on on a large scale
o Protein interac9on
o Immune-pep9domics
o Roughly you can see how proteins are folded
- Which statement Is false
o Top down proteomics does not require trypsina9on -> true
o In plasma, protein concentra9ons differ in concentra9on by 11 orders of
magnitude -> true
o System biology integrated the knowledge of the state and proper9es of alle
proteins -> false
Reverse phase HPLC in proteomics
Introduc)on
- RP: separates molecules on the basis of their reversible interac9on with the hydrofobic
surface of a chromatographic medium
- The proteins are unfolded and thus denatured during the chromatography.
o You can not use it for func9onal studies aKerwards
- Since mid ‘70s
o It is s9ll improving
- Gives nice result, because it has a really good resolu9on
o Peaks of the chromatography are narrow -> peak capacity increases
o The maximum peaks that the chromatography can give you, completely separated
= peak capacity
Hydrophobic interac)ons
- RP-HPLC is mainly based on the reversible hydrophobic interac9ons between amino
acid side chains on the protein and the hydrophobic surface on the chromatographic
medium (sta9onary phase).
o Hydrophobic surface = carbohydrogen chain which has no charge and polarity
- Hydrofobic binding is the result of an entropic effect that has preference:
o Driven by each other by the surrounding water
- Condi9ons at start are usually hydrophilic solvent (e.g. buffered water) such that a high
degree of organized water surrounds the protein surface and sta9onary phase
- Binding of the protein to the sta9onary phase minimizes the amount of exposed
hydrophobic surface on the protein and sta9onary phase:
o surface = 4 πr2 , volume = 3/4 πr3
o Ra9o surf./vol. decreases with increasing r
§ Less organiza9on of water when your molecule a^ach to the hydrophobic
stage compared when to the two are apart
, - This causes reduc9on of water organiza9on and thus corresponding increase in
entropy, crea9ng an energe9c more favourable situa9on for the proteins to associate
with the sta9onary phase.
- The composi9on of the mobile phase is gradually changed as to bring the proteins
differen9ally into the mobile phase (desorp9on).
- A RP-HPLC column is therefore run with an increasing concentra9on of hydrophobic
solvent.
- Proteins are concentrated and purified.
- Get off the sta9onary phase one by one -> called elu9on -> by increasing the
hydrophobicity of the solvent. The proteins that bind to the sta9onary phase can
choose: stay on the sta9onary phase or solve in the solevent
o Proteins not very hydrophobic -> come easy of the sta9onary phase, come in the
mobile phase -> come off
o Proteins very hydrophobic -> come off if the solvent is very hydrophobic
- 215 nm = wavelength at which a pep9de bound absorbers UV light
- Every peak represents a pep9de or a protein
o Acetonitrile is the hydrophobic solvent -> make gradient
o You can gradually elute the protein or pep9de
o Small volume -> quite concentrated -> detec9on very good
Standard condi)ons for RP-HPLC
Sta$onary phase
- Large pressure in the beads
o The smaller the beads the be^er the separa9on is, the higher the number
theore9cal plates the be^er the efficacy of the column is
- Silica-based: mechanically strong, high binding capacity.
o Very strong -> they have high binding capacity
o Strong scaffold
o Can resistant a high pressure
- Beads have pores that increase the ac9ve surface
- Macroporous packing (pores > 300Å) with beads of 5-10 µm diameter most frequently
used.
o Envolving to 5 um or lower -> these beads give the be^er resolu9on
- A^achment of func9onal groups on the beads:
o C4, C8 (octyl) alkyl chains for more hydrophobic analytes
o C18 (octadecyl) alkyl chains for more hydrophilic analytes
§ More hydrophobic
§ You be^er use that for analytes for that a less hydrophobic
- Smaller beads give be^er performance (Van Deemter curve!), however, backpressure
will increase.
o 50s: beads of diameter of 100 um
§ Theore9cal plates were 200
o Now: beads of 1.8 um
§ More theore9cal plate -> much be^er -> peaks are sharper
§ Analy9cal chromatography
- Recent advancement: on-chip liquid chromatography
o 105 - 106 theore9cal plates (50 cm)!
o e.g. etched pillar array chip can be directly coupled to mass spectrometer
Quiz
- List the different aspect of problems where proteomics can give answers to instead of
genomic or transcriptomics
o Alterna9ve splicing
o Protein iden9fica9on on a large scale
o Protein interac9on
o Immune-pep9domics
o Roughly you can see how proteins are folded
- Which statement Is false
o Top down proteomics does not require trypsina9on -> true
o In plasma, protein concentra9ons differ in concentra9on by 11 orders of
magnitude -> true
o System biology integrated the knowledge of the state and proper9es of alle
proteins -> false
Reverse phase HPLC in proteomics
Introduc)on
- RP: separates molecules on the basis of their reversible interac9on with the hydrofobic
surface of a chromatographic medium
- The proteins are unfolded and thus denatured during the chromatography.
o You can not use it for func9onal studies aKerwards
- Since mid ‘70s
o It is s9ll improving
- Gives nice result, because it has a really good resolu9on
o Peaks of the chromatography are narrow -> peak capacity increases
o The maximum peaks that the chromatography can give you, completely separated
= peak capacity
Hydrophobic interac)ons
- RP-HPLC is mainly based on the reversible hydrophobic interac9ons between amino
acid side chains on the protein and the hydrophobic surface on the chromatographic
medium (sta9onary phase).
o Hydrophobic surface = carbohydrogen chain which has no charge and polarity
- Hydrofobic binding is the result of an entropic effect that has preference:
o Driven by each other by the surrounding water
- Condi9ons at start are usually hydrophilic solvent (e.g. buffered water) such that a high
degree of organized water surrounds the protein surface and sta9onary phase
- Binding of the protein to the sta9onary phase minimizes the amount of exposed
hydrophobic surface on the protein and sta9onary phase:
o surface = 4 πr2 , volume = 3/4 πr3
o Ra9o surf./vol. decreases with increasing r
§ Less organiza9on of water when your molecule a^ach to the hydrophobic
stage compared when to the two are apart
, - This causes reduc9on of water organiza9on and thus corresponding increase in
entropy, crea9ng an energe9c more favourable situa9on for the proteins to associate
with the sta9onary phase.
- The composi9on of the mobile phase is gradually changed as to bring the proteins
differen9ally into the mobile phase (desorp9on).
- A RP-HPLC column is therefore run with an increasing concentra9on of hydrophobic
solvent.
- Proteins are concentrated and purified.
- Get off the sta9onary phase one by one -> called elu9on -> by increasing the
hydrophobicity of the solvent. The proteins that bind to the sta9onary phase can
choose: stay on the sta9onary phase or solve in the solevent
o Proteins not very hydrophobic -> come easy of the sta9onary phase, come in the
mobile phase -> come off
o Proteins very hydrophobic -> come off if the solvent is very hydrophobic
- 215 nm = wavelength at which a pep9de bound absorbers UV light
- Every peak represents a pep9de or a protein
o Acetonitrile is the hydrophobic solvent -> make gradient
o You can gradually elute the protein or pep9de
o Small volume -> quite concentrated -> detec9on very good
Standard condi)ons for RP-HPLC
Sta$onary phase
- Large pressure in the beads
o The smaller the beads the be^er the separa9on is, the higher the number
theore9cal plates the be^er the efficacy of the column is
- Silica-based: mechanically strong, high binding capacity.
o Very strong -> they have high binding capacity
o Strong scaffold
o Can resistant a high pressure
- Beads have pores that increase the ac9ve surface
- Macroporous packing (pores > 300Å) with beads of 5-10 µm diameter most frequently
used.
o Envolving to 5 um or lower -> these beads give the be^er resolu9on
- A^achment of func9onal groups on the beads:
o C4, C8 (octyl) alkyl chains for more hydrophobic analytes
o C18 (octadecyl) alkyl chains for more hydrophilic analytes
§ More hydrophobic
§ You be^er use that for analytes for that a less hydrophobic
- Smaller beads give be^er performance (Van Deemter curve!), however, backpressure
will increase.
o 50s: beads of diameter of 100 um
§ Theore9cal plates were 200
o Now: beads of 1.8 um
§ More theore9cal plate -> much be^er -> peaks are sharper
§ Analy9cal chromatography
- Recent advancement: on-chip liquid chromatography
o 105 - 106 theore9cal plates (50 cm)!
o e.g. etched pillar array chip can be directly coupled to mass spectrometer
