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!

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?

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.

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).

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.

Enrichment of Phosphopeptides using TiO2 or IMAC

Non-phosphorylated ‘contaminants’ from IMAC represent abundant rather than acidic peptides

(Figure 7)

Novel Phosphorylation Sites on:

MS/MS spectra for two phosphopeptides

H+-ATPases

Novel Phosphorylation Sites on H+-ATPases

New Sites, Unknown function

Known site, 14-3-3 protein binding site (activates pump).

Fuscicoccin—the Wilting Toxin

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)

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

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?