network and pathway analysis of post-translationally modi ...network and pathway analysis of...
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Jeffrey C. Silva1; Stuart Tugendreich2; Hongbo Gu1; Charles L. Farnsworth1; Kimberly A. Lee1; Xiaoying Jia1; Jian Min Ren1; Matthew P. Stokes1
1Cell Signaling Technology, Danvers, MA USA 01923; 2Ingenuity Systems, Redwood City, CA 94063
Network and Pathway Analysis of Post-translationally Modified Peptides Identified by Immunoaffinity-based Proteomics
Post-translational modification of proteins provides critical regulation of protein activity, stability, and localization in nearly all aspects of cellular signaling. We have developed antibodies that recognize specific post-translational modifications (phosphorylation, ubiquitination, acetylation, methylation), consen-sus kinase substrate motifs, or peptides from proteins that reside in the same signaling pathway or pathways. The antibodies are used to immunoaffinity purify post-translationally modified peptides from cell lines, tissues, xenografts, or other biological materials which are then identified using LC-MS/MS, enabling identification of thousands of post-translationally modified peptides in a single LC-MS/MS run. Ingenuity® Systems pathway analysis software IPA® has been applied to these large datasets in order to facilitate interpretation of the LC-MS/MS data and provide a biological context to the results.
Ingenuity® Systems IPA® software provides in-depth analysis of the complimentary biological networks assembled using the Acetyl-Lysine, Mono-Methyl-Arginine, and Ubiquitin-Branch motif antibodies in PTMScan®.
1. Rush, J. et. al. (2005) Nat. Biotechnol. 23, 94–101.2. Lee, K. A. et. al. (2011) J. Biol. Chem. 286(48):41530–41538.
Introduction
Conclusion References
Contact InformationJeffrey C. Silva, Cell Signaling Technology, Inc. Email: [email protected]
PTMScan® Services Department Email: [email protected] • web: www.cellsignal.com/services© 2013 Cell Signaling Technology, Inc. / Cell Signaling Technology®, PhosphoScan®, PhosphoSitePlus®, PTMScan®, AcetylScan®, UbiScan®, and MethylScan™ are trademarks of Cell Signaling Technology, Inc. / LTQ-Orbitrap® is a registered trademark of THermo Fisher Scientific. / IPA® is a registered trademark of Ingenuity Systems, Inc. / Sorcerer™ is a trademark of Sage-N Research.
A B
C
MethodsPTMScan® analysis (Rush et al. 2005, Lee et al. 2011, Kim et al. 2011) was performed on HCT-116 human colon carcinoma and mouse liver tis-sue peptides with the Acetyl-Lysine, Mono-Methyl-Arginine, or Ubiquitin Branch Antibodies from Cell Signaling Technology. Samples were run on LTQ-Orbitrap VELOS™ or Elite LC-MS/MS instruments and searches were performed using Sorcerer™ software (Lundgren et al. 2009). Results from each study were combined and imported into the Ingenuity® Systems IPA® software package. Core analyses were run on all proteins identified as well as proteins unique to each anti-body using the Ingenuity Knowledge Base of genes and endogenous chemicals limited to direct interactions with experimental and high confidence predicted relationships. Figure 1: PTMScan Method: Immunoaffinity purification workflow using
motif antibodies for enrichment of acetylated (AcetylScan®), methylated (MethylScan™), and ubiquitinated (UbiScan®) peptides.
Figure 2: PTMScan® results using Acetyl-lysine, Mono-methyl-Arginine, and Ubiquitin Branch motif antibodies. A. Number of non-redundant sites/proteins identified for each antibody. B. Area proportional Venn diagram showing the number of proteins identified using each anti-body (black numbers) and overlap between antibodies (white numbers). C. Top seven most highly represented protein classes identified with each antibody. Protein type information from the PhosphoSitePlus® database.
Figure 3: A. Bar graphs of the highest scoring canonical pathways for each antibody. B-D. Selected canonical IPA® Signaling Pathways are color-coded for each antibody (Acetyl = Red, Methyl = Blue, Ubiquitin = Green). A composite pathway is shown for each (Yellow highlights).
Figure 4: The top three IPA®-generated networks are shown for each antibody (A = Acetyl, B = Methyl, C =Ubiquitin). The overlap of the networks created for each antibody are shown with the selected networks displayed in detail.
Acetyl Methyl Ubiquitin
Sites 10,191 3,058 14,977
Proteins 3,195 1,099 4,453
Ubiquitin
Methyl
1099
31954453 1030
164192 100
Acetyl
11.2
1.4
12.8
7.7
6.7
12.3
13.4
4.6
0.2
6.0
16.5
38.9
1.9
3.3
6.6
15.0
8.2
7.2
2.6
16.1
25.7
0.0 10.0 20.0 30.0 40.0
Receptor/Transporter/Cell Surface
Mitochondrial Protein
Adhesion/Extracellular
Transcription
RNA Processing
Enzyme
DNA Binding/Repair/Replication
Percent of Total
AcetylMethylUbiquitin
IPA® Canonical Pathways: Mapping identified proteins onto pre-defined signaling networks
IPA®-Generated Networks: De novo pathway assembly from identified proteins
A B C D
Acetyl Methyl
Ubiquitin
A Acetyl B Methyl
3. Kim, W. et. al. (2011) Mol. Cell. 2011 Oct 21;44(2):325–340.4. Lundgren, D. H. et. al. (2009) Curr. Protoc. Bioinformatics Chapter 13, Unit 13 13.
PI3K-Akt Signaling
Acetyl
Methyl
Ubiquitin
Mouse Embryonic Stem Cell Pluripotency
Acetyl
Methyl
Ubiquitin
Molecular Mechanisms of Cancer
Acetyl
Methyl
Ubiquitin
C Ubiquitin