1. I.Introduction to PTMs II.Protein Phosphorylation (Ser/Thr) A.Methods―enrichment. B.Applications i.Membrane “shaving” ii.Label-free quantitative analysis iii.Large-scale phosphorylation site mapping iv.pS/pT-binding proteins (intro to Pro:Pro) Lecture 9 Inclusive Illinois Day- one campus, many voices...many PTMs!

2. Protein Phosphorylation One of the most common intracellular protein modification Approximately 5 % of the Arabidopsis thaliana genome encodes for protein kinases & phosphatases (H. sapiens: 5 %) Approximately 30 % of all human proteins are phosphorylated. How about plants, e.g., A. thaliana? Protein phosphorylation is of special importance in the regulation of functions, e.g. metabolism or cell signaling In order to better understand the molecular mechanisms where phosphorylation is involved this modification has to be analyzed. So why do you need enrichment?

3. 1)Ionization suppression Phosphorylation Analysis by MS – The Problems Stensballe et al., Proteomics. 2001, 1, 207. Phosphopeptides do not fly well and their ionization can be suppressed by nearby (more abundant) ions. Also: low stoichiometry and loss due to phosphatase action.

4. Mol. Cell Proteomics 2 (2003): 1234-1243. “MEMBRANE SHAVING” A.Use of Brij-58 to invert PM vesicles as demonstrated by ATP-dependent proton pumping. B.Bacterial virulence factors (flg22) increase in vivo phosphorylation of PM proteins (upward arrows; TLC peptide mapping).

5. In Vivo Phosphorylated Membrane Proteins 2-dimensional LC separation of proteins. Enrichment of phosphopeptides by IMAC step is apparent. Multiply phosphorylated peptides tend to elute at high salt, as expected.

6. Enrichment of Phosphopeptides using TiO 2 or IMAC

7. Non-phosphorylated ‘contaminants’ from IMAC represent abundant rather than acidic peptides (Figure 7)

8. Novel Phosphorylation Sites on: MS/MS spectra for two phosphopeptides H + -ATPases

9. Novel Phosphorylation Sites on H + -ATPases New Sites, Unknown function Known site, 14-3-3 protein binding site (activates pump).

10. Fuscicoccin—the Wilting Toxin

11. MS-Based Phosphorylation Analysis: 1)Enrichment of phosphorylated proteins/peptides Anti-phosphoamino acid antibodies IMAC Derivatization 2) localization of the phosphorylation site MALDI-ToF-MS LC-MS/MS (e.g., LTQ-Orbitrap)

12. Chemical derivatization Introduce affinity tag to enrich for phosphorylated molecules e.g., biotin binding to immobilized avidin/streptavidin Oda et al., Nature Biotech. 2001, 19, 379 for analysis of pS and pT Enrichment strategies to analyze phosphoproteins/peptides

13. 1 MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE 51 KVNELSKDIG SESTEDQAME DIKEMEAESI SSSGEIVPNS VEQKHIQKED 101 VPSERYLGYL EQLLRLKKYK VPQLEIVPNS AEERLHSMKE GIDAQQKEPM 151 IGVNQELAYF YPELFRQFYQ LDAYPSGAWY YVPLGTQYTD APSFSDIPNP 201 IGSENSEKTT MPLW 1 MKLLILTCLV AVALARPKQP IKHQGLPQGV LNENLLRFFV APFPEVFGKE 51 KVNELSTDIG SESTEDQAME DIKQMEAESI SSSEEIVPIS VEQKHIQKED 101 VPSERYLGYL EQLLRLKKYN VPQLEIVPNL AEEQLHSMKE GIHAQQKEPM 151 IGVNQELAYF YPQLFRQFYQ LDAYPSGAWY YVPLGTQYPD APSFSDIPNP 201 IGSENSEKTT MPLW Casein alpha S1 Alpha-S1 casein HOMEWORK II—MS/MS Database Searching 4. One of the proteins is not phosphorylated in vivo. T or F?