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PTMs, Degradomics, Phosphoproteomics - Coggle Diagram

- - - - irreversible
    - - reversible
    - - reversible
    - - irreversible
    - - reversible
- - - - Digest proteins to peptides
        
        Fractionation
        
        LC/MS
        
        Data analysis
        
        Reduces sample complexity by separating peptides before MS
        
        Chromatographic separation, such as SCX (Strong cation Exchange) or HILIC
        
        Enrichment for PTMs through Ab/metal chromatography
      - Enrichment for PTMs through Ab
- - - - Aminopeptidases
      - Carboxypeptidases
      - Endopeptidases
      - The mechanism of action also allows us
        to classify them into families
        
        Aspartyl proteases
        
        Asp residues in the catalytic site
        
        Cysteine proteases
        
        Cys residue in the catalytic site
        
        Metalloproteases
        
        The residues in their catalytic site coordinate metal ions, such as Zn2+
        
        Serine proteases
        
        Ser residue in the catalytic site
        
        Threonine proteases
        
        Thr in the catalytic site
  - - - pH-dependent activation
      - Localization
      - Cofactors/allosteric activation
      - Inhibitors are also available
        
        For example, Covid treatments block the proteases involved in the cleavage of the viral polyprotein
- - - - Enrichment
        
        Negative
        
        Removes all abundant, unwanted internal peptides (generated by trypsin cleavage) while leaving the desired N-terminal peptides untouched
        
        TAILS (Terminal Amine Isotopic Labeling of Substrates)
        
        Involves an iTRAQ-like NH2-labeling of primary amines before trypsin digestion. Trypsin cleavage will generate free N-terminals, which will be bound to an amine-reactive HPG-ALD polymer and precipitated. Instead, N-terminals generated by proteases will be labelled and will be retained.
        
        1 more item...
        
        Important: cleavage with trypsin occurs after cleavage with protease
        
        Positive
        
        Directly captures the target N-terminal peptides
        
        PICS (Proteomic Identiﬁcation of protease Cleavage Sites)
        
        After trypsin digestion, the N-terminals are chemically blocked. Subsequently, the peptides are incubated with the protease of interest, which will generate new neo N-terminals. Neo N-terminals will be biotinylated and isolated using streptavidin in a pull down assay.
        
        Important: cleavage with trypsin occurs before cleavage with protease
- - - - Cleavage with protease of interest
        
        Trypsin cleavage
        
        MS analysis
- - - - 1st LC
        
        Enrichment
        
        Fractionation
        
        LC/MS
        
        Fundamental step because the % of phosphorylated proteins in a cell is low
        
        Different techniques:
        
        P-Tyr
        
        Ab
        
        P-Ser, P-Thr
        
        Binding to positively charged metal ions or oxides (Fe3+, TiO2)