Dit document omvat de info van de slides plus mijn lesnotities van les 5 van concepts, gegeven door Xaveer Van Ostade. De lessen van Kurt Boonen (Les 2,3,4 en 8) heb ik ook, maar de notities staan op de slides. Moest je hierin ook geïnteresseerd zijn, mag je me altijd contacteren via messenger :))
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
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