1. MN-B-C 2 Analysis of High Dimensional (-omics) Data Kay Hofmann – Protein Evolution Group http://www.genetik.uni-koeln.de/groups/Hofmann Week 5: Proteomics 2

2. Irreversible: Proteolytic Protein Processing  Many newly synthesized proteins contain portions that are not required for the ultimate protein function but server other purposes.  Various 'signal sequences' contain localization information for the protein and are removed once the final destination has been reached.  Some proteins - mostly enzymes - are synthesized as inactive pro-proteins, e.g. to avoid damage by acting at the wrong place.The 'pro-sequence' is proteolytically removed once the destination is reached. The pro-form can also act as storage form that gets activated on demand. Irreversible: Proteolytic Degradation  Many proteins are degraded when they are defective or no longer needed. Different degradation systems exist inside and outside of cells. Protein degradation if typically a highly regulated process. (Mostly) Irreversible: Protein Glycosylation  Lumenal portions of proteins are often glycosylated in a multi-step reaction during passage through ER and Golgi. Functions of glycosylation are diverse, often not understood (N-Glycosylation →Asn, O-Glycosylation → Ser/Thr)

3. Purpose  Many PTMs are reversible and regulate various aspects of protein function  Mode #1: Modification directly changes protein conformation/activity  Mode #2: Modification changes protein interaction, e.g. through specific recognition factors for the modified residue (or for the unmodified residue).  Mode #3: Modifications can also regulate the stability of a protein or enhance/prevent other modifications.  Both on- and off-reactions are typically highly regulated processes. Overview  Phosphorylation ( on Ser, Thr or Tyr, rarely on His)  Ubiquitination (on Lys, rarely on N-terminus)  Sumoylation and other UBL-Modifications (on Lys)  Acetylation (on N-terminus or Lys)  Methylation (on Lys or Arg)  Lipidation (on Cys or protein termini)  Nitrosylation (on Cys)

4. The three amino acids with Hydroxyl-Groups can form phosphate-esters. The reaction is catalysed by so-called protein kinases (under consumption of ATG). Phosphate groups can be hydrolytically removed by protein phosphataseas. Mainly in bacteria, a system for the phosphorylation of His-residues is typical. Abb.: Alberts

5. Protein Phosphorylation can change the properties/activity of the target protein. Phosphorylated proteins are recognized by specialized binding domains (e.g. SH2 for phospho-Tyr, FHA for phospho-Ser/Thr) Humans have about 500 different protein kinases and about 120 different phosphatases. Regulation by phosphorylation is widespread in signal transduction, e.g. through the use of 'kinase cascades'.

6. kinase -P-P Ras SH2 SH3 Ras GEF Pro kinase RBD kinase EGF-ReceptorGrb2 SOS (rasGEF) RasRafKinase cascade GTP EGF membrane kinase -P-P Cytokine- Receptor Cytokine membrane SH2 DNA-bind NucleusDNA JAK kinase STATGene regulation Pathway based on phosphorylation and specific recognition of phospho-sites. Many of these pathway contain kinase cascades.

7. Ubiquitin ist a small protein (76 residues), whose C-terminus can (in a three-step procedure) be covalently coupled to Lysine-NH2 Groups in the target protein. Abb.: Stryer

8. Unlike phosphorlytion, ubiquitination rarely/never leads to a direct activity change of the target protein. Ubiquitinated proteins are recognized by specialized binding proteins or domains (UBA, UIM, UBZ). Some binding partners require a particular chain, others are substrate-specific. Humans have about 40 E2 and 500 E3 enzymes (Ubiquitin Ligases) and about 100 deubiquitinases (DUBs). Die E2 determines the chain type, the E3 determines the substrate. Ubiquitin itself contains several lysine residues that can be ubiquitinated. The resulting chain types can form different signals (e.g. chain of 4 ubiquitins attached via Lys-48 leads to degradation)

9. Besides ubiquitin, there are 12 more related proteins, many of which can be activated and conjugated onto proteins by a mechanism analogous to ubiquitin. The enzymes involved in these pathways are different from those involved in ubiquitination, but are related to them. SUMO regulates nuclear import/export and the formation of 'nuclear bodies'. NEDD8 regulates a large class of ubiquitin ligases Atg8 regulates autophagy.

10. Motivation 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.

11. Procedure Only interested in modified peptides - no need to waste MS resources on unmodified peptides. For overview studies: 1) (optional) enrichment of proteins carrying the desired modification (e.g. antibodies) 2) digestion 3) enrichment of peptides carrying the desired modification (antibodies, columns) 4) 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. ----------K--------K------K---------- Ubiquitin-K-G-G

12. Phospho-Tyr can be recognized by antibody SCX=strong cation exchange IMAC= immobilized metal affinity chromatography

13. Demonstration of Phosphosite Plus (http://www.phosphosite.org) ELM (http://elm.eu.org)