MN-B-C 2 Analysis of High Dimensional (-omics) Data. Week 5: Proteomics 2. Kay Hofmann – Protein Evolution Group http://www.genetik.uni-koeln.de/groups/Hofmann. Posttranslational Modifications (PTMs). Irreversible: Proteolytic Protein Processing
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MN-B-C 2 Analysis of High Dimensional (-omics) Data
Week 5: Proteomics 2
Kay Hofmann – Protein Evolution Grouphttp://www.genetik.uni-koeln.de/groups/Hofmann
Irreversible: Proteolytic Protein Processing
(Mostly) Irreversible: Protein Glycosylation
Irreversible: Proteolytic Degradation
The threeaminoacidswithHydroxyl-Groups can form phosphate-esters. The reactioniscatalysedby so-calledprotein kinases (underconsumptionof ATG). Phosphate groupscanbehydrolyticallyremovedbyproteinphosphataseas.
Mainly in bacteria, a systemforthe phosphorylation of His-residuesistypical.
Protein Phosphorylation canchangetheproperties/activityofthetargetprotein.
Phosphorylated proteinsarerecognizedbyspecializedbindingdomains (e.g. SH2 forphospho-Tyr, FHA forphospho-Ser/Thr)
Humanshaveabout500 different protein kinases andabout120 different phosphatases.
Regulation by phosphorylation iswidespread in signaltransduction, e.g. throughtheuseof 'kinase cascades'.
Pathway based on phosphorylation and specific recognition of phospho-sites.
Many of these pathway contain kinase cascades.
Ubiquitin ist a smallprotein (76 residues), whoseC-terminuscan(in a three-stepprocedure) becovalentlycoupledtoLysine-NH2 Groups in thetargetprotein.
Ubiquitin itselfcontainsseverallysineresiduesthatcanbe ubiquitinated. The resultingchaintypescan form different signals(e.g. chainof 4 ubiquitinsattached via Lys-48 leadstodegradation)
Humanshaveabout40 E2 and500 E3 enzymes(Ubiquitin Ligases) andabout 100 deubiquitinases(DUBs). Die E2 determinesthechain type, the E3 determinesthesubstrate.
(UBA, UIM, UBZ). Somebindingpartnersrequire a particularchain, othersaresubstrate-specific.
Unlikephosphorlytion, ubiquitination rarely/neverleadsto a directactivitychangeofthetargetprotein.
Besides ubiquitin, thereare 12 morerelatedproteins, manyofwhichcanbeactivatedandconjugatedontoproteinsby a mechanismanalogousto ubiquitin. The enzymesinvolved in thesepathwaysare different fromthoseinvolved in ubiquitination, but arerelatedtothem.
SUMO regulatesnuclearimport/exportandtheformationof 'nuclearbodies'.
NEDD8 regulates a large classof ubiquitin ligases
Atg8 regulates autophagy.
Modification proteomics begins with simple questions like e.g. which proteins can be modified by phosphorylation/ubiquitination, which sites are affected, is there a 'site consensus', etc.
The large number of protein kinases and ubiquitin ligases (~500 each) and the somewhat smaller number of phosphatases and deubiquitinases (~100 each) begs the question for substrate specificity.
Task: which are the targets of kinase/ligase X ?
Task: which kinase/ligase acts on substrate Y?
Since phosphorylation and ubiquitination have important roles in signal transduction, other typical questions are:
Task: which substrates get phosphorylated/ubiquitinated in cell type X stimulated by Y.
Only interested in modified peptides - no need to waste MS resources on unmodified peptides. For overview studies:
(optional) enrichment of proteins carrying the desired modification (e.g. antibodies)
enrichment of peptides carrying the desired modification (antibodies, columns)
tandem MS, spectral counting.
Example: ubiquinated proteins can be enriched by affinity purification with an anti-ubiquitin antibody (if necessary: linkage-specific).
After digestion with Trypsin, each ubiquinated peptide will contain a lysine residue that is covalently modified to a Gly-Gly dipeptide (via iso-peptide bond at the -NH2 group)
Finally, the peptides containing the Gly-Gly stub can be enriched by a recently devolped antibody directed at Gly-Gly-modified Lysine.