Proteomics

enrichment to focus our measurement on the sub proteome of interest

• enrichment of phosphopeptides
  • affinity enrichement: IMAC: immobilized metal affinity chromatography -> affinity of phospho groups toward metal ions (Fe3+)
  • antibody based: ab that is directed against certain PTM (immuno affinity method)
• enrichment of ubiquitination
  • antibody based: ab specific for the di-glycyl moiety present on the side chains of ubiquitinated lysine residues after trypsin digestion

Mass spec

Phosphorylation: neutral loss of 97.97

MS1: peptide mass increased by mass of the PTM

MS2: mass of some fragments shifted by ∆m => depending on where P is, it can be seen on diff. fragments

scoring system tells us which localization is more probable

Ubiquitination: K-e-GG shows no neutral loss

∆m: Lyss + 2x Gly = 242.1

fish for interactions using an affinity ligand

to consider: 

• specific vs unspecific binding: not all proteins bind specifically -> experimental setup need to distinguish
• interaction dilemma: in affinity purification, mostly proteins with high abundance &/or affinity/residence time seen
• protein abundance: required for affinity purification: high recovery, fast purification methods, high sensitivity analytics (to also see low abundant complexes)
• biochemical realities: affinity & kinetics are affected by experimental conditions
• timing:

• Antibody - targeted: co-immunoprecipitation: AB against POI, pulldown of complex, enrichment of bait protein and interacting proteins
  • pro: no cloning, fast
  • con: not generic, cross reactivity
• antibody - generic: epitope tagging: POI genetically fused to epitope tag
  • pro: generic, highly reproducible
  • con: tag may influene protein expression
• Tandem affinity purification TAP: POI with dual affinity tag; TEV: cleavage site for TEV protease, BCBP calmodulin binding protein. elution of bait by EGTA
  • pro: generic, highly reproducible
  • con: tag might influence protein function

• Peak capacity describes the maximum theoretical number of analytes that can be succesfully seperated with a given column and a set of analytical paramenters.
• It depends on chromatographic resolution and gradient time. 
• In practice: highest peak capacities are obtained with long efficient columns and long gradient times