![Page 1: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/1.jpg)
Protein Correlation Profiling for Label-Free Quantitation
![Page 2: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/2.jpg)
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!
![Page 3: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/3.jpg)
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)
![Page 4: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/4.jpg)
Sucrose Supply Increases Phosphorylation of Thr-947 Activating Proton Pumping
![Page 5: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/5.jpg)
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
![Page 6: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/6.jpg)
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
![Page 7: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/7.jpg)
Posttranslational Modifications—II
Protein:Protein Interactions
Genome
Proteome
Interactome
PTMs
![Page 8: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/8.jpg)
All interactions Interaction network of disease-associated proteins.
![Page 9: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/9.jpg)
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.
![Page 10: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/10.jpg)
A membrane protein/signaling protein interaction network for Arabidopsis version AMPv2 Sylvie Lalonde1*, X(22) and Wolf B. Frommer1
Frontiers in Plant Physiology
![Page 11: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/11.jpg)
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
![Page 12: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/12.jpg)
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
![Page 13: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/13.jpg)
Protein Interaction Domains
Pro-rich binding: pY binding:
“general”pS/pT binding:
Phospholipid binding:
![Page 14: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/14.jpg)
Protein Interaction Domains
![Page 15: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/15.jpg)
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).
![Page 16: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/16.jpg)
14-3-3 Modes of Action: illustrative examples(Tzivion et al. JBC 277 (2002) 3061-3064)
Molecular ‘Anvil’ mechanism for 1433s
![Page 17: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/17.jpg)
Identification of 14-3-3-binding proteins in vitro
![Page 18: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/18.jpg)
![Page 19: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/19.jpg)
![Page 20: Protein Correlation Profiling for Label-Free Quantitation](https://reader036.vdocuments.site/reader036/viewer/2022062518/56649ea65503460f94ba93c3/html5/thumbnails/20.jpg)
14-3-3-binding proteins (‘clients’) in Barley