Sample preparation

Prof. Boonen - Ma 1 BMW ('23-'24) - concepts of protein technology

Alternative splicing gives rise to

different isoforms

What are proteoforms?

different versions of the same protein arising from SNPs, PTM, differential splicing

What is the protein interference problem?

Difficult to know which proteins/proteoforms were in a complex sample once digested

What are the different steps in the sample preparation?

1. define the research question

2. literature study

3. sample collection

4. protein extraction + solubilisation

5. (protein separation / purification)

6. reduction + alkylation

7. digestion

8. peptides purification / selection

9. (prefractionation)

How can you minimise sample degradation?

• inhibitors (protease and phosphatase)

• denaturation (chaotropic agents + detergents)

• temperature adjustment

• pH adjustment

What are the different techniques for protein extraction? Result?

Mechanical vs non mechanical

Soft vs harsh

Soft

• osmotic shock: expose cells to low/high salt concentration and switch to hig/low salt concentration → osmotic pressure → cell lysis

• freezing: repeated cycles of freezing and thawing → disrupt cells through ice crystal formation

• detergents: break membranes and solubilise proteins

• enzymatic digestion

• homogeniser: cells between glas rod and inside of glass tube

Harsh

• blender

• tissue chopper

• cryo-grinding: use pestle and mortar under liquid N₂

• bead beater

• sonication: pulsed high frequency sound waves - in ice bath

→ sample with proteins in solution + remnants of cells → subcellular fractionation: differential centrifugation or sucrose gradient

How do you solubilise proteins?

! chose optimal lysis buffer for extraction !

Denaturing vs non-denaturing

Chaotropic agents

• = disruption of non-covalent bonds

• denaturing

• change H bonds in solvent + disrupt H bonds in proteins

• lower energy barrier for exposing apolar groups

• urea: 2D PAGE, H bond disruption!, hydrophobic disruption

• thio-urea: hydrophobic disruption, membrane proteins

• guanidinium chloride: H

Detergents

• form micelles with hydrophobic core (→ bind proteins) and hydrophilic outside

• denaturing: SDS, ETMAB → cooperative, binding of 1 SDS will increase chance of another SDS bidning to molecule, protein becomes rigid rod proportional to MW

• non denaturing

  • nonionic: tritonX100, tween → protein function and interactions remain, break lipid-lipid and lipid-protein interactions

  • zwitterionic: CHAPS → disrupt protein-protein interactions, isoelectric focussing + ion-exchange chromatography

Why do detergents need to be removed?

Interfere with analysis

Use: dialysis, gel filtration chromatography, cut-off filter, protein precipitation, SDS-PAGE

What is the function of reduction and alkylation?

Breaking of disulfide bridges

Add alkylgroup to stabilise reduced cystein

• beta-mercaptoethanol

• DTT: dithiothreitol

Why use carboxymethylation and carbamidomethylation?

To prepare proteins for digestion in LC-MS protocols, prior to sequencing

• enhances proteolytic digestion

• ! mass increases

What are the options to detect low abundant proteins?

High abundant protein depletion → antibodies

Low abundant protein enrichment = concentrate on specific affinity ligands

How are proteins digested?

Trypsin

• result: peptides of 10-20 AA

• cleaves after Lysine (K) or Arginine (R)

→ optional labeling to primary amines

How do you clean up peptide mix?

C18 solid phase extraction

• peptides bind to beads = hydrophobic

• elution by acetonitrole

• larger peptides stay on column

Ion exchange chromatography: strong cation exchange

• acid condition: peptides are + charged

Why use prefractionation?

additional liquid chromatography step → reach more depth