Protein Correlation Profiling for Label-Free Quantitation

Proteome Coverage for Quantitation

Only a subset of the proteome is identified, and of that, only the higher abundance proteins can be quantitated!

Only a subset of the proteome is identified, and of that, only the higher abundance proteins can be quantitated!

Arabidopsis Seedlings: LL + Sucrose , 7 days

DD - Sucrose, 1 day

Resupply 30 mM Suc in the dark: 0 to 30 min

Plasma Membranes isolated

Phosphpeptides enriched (IMAC)

LC/MS/MS and MS3 (label free quantitation)

Sucrose Supply Increases Phosphorylation of Thr-947 Activating Proton Pumping

850 860 870 880 890 900

IRYILSGKAW ASLFDNRTAF TTKKDYGIGE REAQWAQAQR TLHGLQPKED VNIFPEKGSY

910 920 930 940

RELSEIAEQA KRRAEIARLR ELHTLKGHVE SVAKLKGLDI DTAGHHYTV

Novel Regulation of the H+-ATPase by C-terminal Phosphorylation

Phospho Thr-881 activates in the absence of a 14-3-3 protein

Phospho Thr-948 activates but only in the presence of a 14-3-3 protein

Clustering of time responses of the quantified phosphorylation sites by increase in sum of squares (Ward's method) reveals four significant response clusters as determined by

greatest deviation from clusters in randomized datasets.Graphs show the mean response curve of all members of that cluster.

Niittylä T et al. Mol Cell Proteomics 2007;6:1711-1726©2007 by American Society for Biochemistry and Molecular Biology

Posttranslational Modifications—II

Protein:Protein Interactions

Genome

Proteome

Interactome

PTMs

All interactions Interaction network of disease-associated proteins.

A Predicted Interactome for ArabidopsisJane Geisler-Lee, Nicholas O'Toole2, Ron Ammar2, Nicholas J. Provart, A. Harvey Millar and Matt Geisler

Figure 2. Visualizing the Arabidopsis predicted interactome. A, Giant hairy ball of all 19,979 interactions visualized by Cytoscape. B, Enlargement showing example of some detail captured by visualization. C, Different types of protein nodes classified as major hubs when interacting with 50 to 100 other proteins, medium hubs 11 to 50, minor hub three to five, pipes two, free end one, and unconnected zero interacting proteins. D, Frequency distribution of different node classes based on number of interacting partners.

A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2 Sylvie Lalonde1*, X(22) and Wolf B. Frommer1

Frontiers in Plant Physiology

Figure 5. Sub-network of receptor kinase interactions (RLK). Labeling as in Figure 3. The blue borders surrounding 31 individual RLKs indicate evidence for phosphorylated sites according to PhosPhAt 3.0 (Durek et al., 2010).

Receptor-Like Kinase Interactions

Figure 8. Ammonium transporter sub-network. (A) AMT1;1 sub-network (AT4G13510). Labeling as in Figure 5. (B) Independent analysis of AMT1;1 interaction with two RLKs identified in AMPv2 using the split luciferase assay in Arabidopsis leaf protoplasts. Red asterisks in the diagram to the right indicate the position of the split luciferase fusions.

Ammonium Transporter

Some Protein Interaction Domains

The figure shows the SH2 domain of v-src bound to a pYEEI peptide ligand.

Phosphotyrosine binding (PTB) domains are 100-150 residue modules that commonly bind Asn-Pro-X-Tyr motifs.

The figure presents the first FHA domain of Rad53 in complex with a phosphothreonine containing peptide. In this structure, the primary contact on the peptide occurs at the phosphothreonine

and the +4 aspartic acid residue.

Dr. Tony PawsonMount Sinai Hospital

Protein Interaction Domains

Pro-rich binding: pY binding:

“general”pS/pT binding:

Phospholipid binding:

Protein Interaction Domains

14-3-3s are highly conserved pSer/pThr-binding proteins(Sehnke, DeLille and Ferl. Plant Cell, Supplement 2002: S339-S354)

Various approaches to study at the 'proteomic level' (Far-Western overlay; immobilized 1433 columns).

14-3-3 Modes of Action: illustrative examples(Tzivion et al. JBC 277 (2002) 3061-3064)

Molecular ‘Anvil’ mechanism for 1433s

Identification of 14-3-3-binding proteins in vitro

14-3-3-binding proteins (‘clients’) in Barley