Network and Pathway Analysis of Post-translationally Modified Peptides
Identified by Immunoaffinity-based Proteomics
Jeffrey C. Silva1; Stuart Tugendreich2; Hongbo Gu1; Charles L. Farnsworth1; Kimberly A. Lee1; Xiaoying Jia1; Jian Min Ren1; Matthew P. Stokes1
1
Introduction
Cell Signaling Technology, Danvers, MA USA 01923; 2Ingenuity Systems, Redwood City, CA 94063
IPA® Canonical Pathways: Mapping identified proteins onto pre-defined signaling networks
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), consensus 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.
A
Acetyl
Methyl
Methods
Ubiquitin
PTMScan analysis (Rush et al. 2005, Lee et al. 2011, Kim et al. 2011)
was performed on HCT-116 human colon carcinoma and mouse liver tissue 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 antibody using the Ingenuity Knowledge Base of genes and endogenous
chemicals limited to direct interactions with experimental and high
confidence predicted relationships.
Molecular Mechanisms of Cancer
B
®
Acetyl
Acetyl
Methyl
Methyl
Ubiquitin
Ubiquitin
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).
IPA®-Generated Networks: De novo pathway assembly from identified proteins
A
A
PI3K-Akt Signaling
D
Acetyl
Methyl
Ubiquitin
Figure 1: PTMScan Method: Immunoaffinity purification workflow using
motif antibodies for enrichment of acetylated (AcetylScan®), methylated
(MethylScan™), and ubiquitinated (UbiScan®) peptides.
Mouse Embryonic Stem Cell Pluripotency
C
Acetyl
B
Methyl
C
Ubiquitin
B
Sites
Proteins
Acetyl
10,191
3,195
Methyl
3,058
1,099
Ubiquitin
14,977
4,453
Ubiquitin
Acetyl
4453
164
C
192
DNA Binding/Repair/Replication
Enzyme
16.1
Ubiquitin
12.3
2.6
7.2
6.0
0.0
Methyl
8.2
12.8
15.0
0.2
1.4
Receptor/Transporter/Cell Surface
1099
16.5
7.7
Adhesion/Extracellular
100
38.9
6.7
Transcription
Acetyl
Methyl
13.4
1.9
RNA Processing
Mitochondrial Protein
25.7
3.3
3195
1030
6.6
4.6
11.2
10.0
20.0
30.0
Percent of Total
40.0
Figure 2: PTMScan® results using Acetyl-lysine, Mono-methyl-Arginine, and Ubiquitin Branch motif antibodies. A. Number of nonredundant sites/proteins identified for each antibody. B. Area proportional Venn diagram showing the number of proteins identified using each antibody (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 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.
Conclusion
References
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.
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.
Contact Information
© 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.
Jeffrey C. Silva, Cell Signaling Technology, Inc. Email: jsilva@cellsignal.com
PTMScan® Services Department Email: ptmscan@cellsignal.com • web: www.cellsignal.com/services