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: jsilva@cellsignal.com

PTMScan® Services Department Email: ptmscan@cellsignal.com • 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