Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jul 15:124:89-99.
doi: 10.1016/j.ymeth.2017.06.017. Epub 2017 Jun 24.

Protein network construction using reverse phase protein array data

Rency S Varghese  1 , Yiming Zuo  2 , Yi Zhao  3 , Yong-Wei Zhang  1 , Sandra A Jablonski  1 , Mariaelena Pierobon  4 , Emanuel F Petricoin  4 , Habtom W Ressom  5 , Louis M Weiner  6
Affiliations

Protein network construction using reverse phase protein array data

Rency S Varghese et al. Methods. .

Abstract

In this paper, we introduce a novel computational method for constructing protein networks based on reverse phase protein array (RPPA) data to identify complex patterns in protein signaling. The method is applied to phosphoproteomic profiles of basal expression and activation/phosphorylation of 76 key signaling proteins in three breast cancer cell lines (MCF7, LCC1, and LCC9). Temporal RPPA data are acquired at 48h, 96h, and 144h after knocking down four genes in separate experiments. These genes are selected from a previous study as important determinants for breast cancer survival. Interaction networks are constructed by analyzing the expression levels of protein pairs using a multivariate analysis of variance model. A new scoring criterion is introduced to determine relevant protein pairs. Through a network topology based analysis, we search for wiring patterns to identify key proteins that are associated with significant changes in expression levels across various experimental conditions.

Keywords: Breast cancer; MANOVA; Network construction; RPPA; Topology analysis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
An overview of the workflow for RPPA data analysis.
Figure 2
Figure 2
A: a schematic representation of the protein pair analysis using MANOVA. The expression level of each protein pair is represented as a bivariate input to the MANOVA model. B: the construction of protein networks for each group comparison. The top scoring protein pairs that are significantly selected in MANOVA analysis and pair-wise analysis are used to construct the protein networks.
Figure 3
Figure 3
Heatmap of changes in protein expression for siRNA-transfected MCF7, LCC1, and LCC9 cells at 48h, 96h and 144h for TOB1, POLR2B, PSMC5, and CYR61 knock down genes using the corresponding negative controls (siNEG) as references.
Figure 4
Figure 4
Protein networks constructed using significant protein-pairs (TOB1 knockdown vs. negative control) for each cell line and each time point.
Figure 5
Figure 5
A: Combined network with most varying nodes and edge changes highlighted for MCF7 vs. LCC1-LCC9. B: Differential network for the most re-wired nodes showing the edge changes in MCF7 vs. LCC1-LCC9.
Figure 6
Figure 6
P21 positively correlated with cyclin D1 after TOB1 knockdown. 20 nM siRNAs against negative, p21 or TOB1 were transfected in LCC1 cells for 48 h and 96 h. Western blot was used to detect protein expression.
See this image and copyright information in PMC

References

    1. Guo H, Zheng Y, Wang B, Li Z. A note on an improved self-healing group key distribution scheme. Sensors (Basel) 2015;15(10):25033–38. - PMC - PubMed
    1. Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, Emmert-Buck MR, Roth MJ, Petricoin EF, III, Liotta LA. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene. 2001;20(16):1981–1989. - PubMed
    1. Eichner J, Heubach Y, Ruff M, Kohlhof H, Strobl S, Mayer B, Pawlak M, Templin MF, Zell A. RPPApipe: A pipeline for the analysis of reverse-phase protein array data. BioSystems. 2014;122:19–24. - PubMed
    1. Daemen A, Griffith OL, Heiser LM, Wang NJ, Enache OM, Sanborn Z, Pepin F, Durinck S, Korkola JE, Griffith M, Hur JS, Huh N, Chung J, Cope L, Fackler MJ, Umbricht C, Sukumar S, Seth P, Sukhatme VP, Jakkula LR, Lu Y, Mills GB, Cho RJ, Collisson EA, van’t Veer LJ, Spellman PT, Gray JW. Modeling precision treatment of breast cancer. Genome Biol. 2013;14(10):R110. - PMC - PubMed
    1. Toettcher JE, Weiner OD, Lim WA. Using optogenetics to interrogate the dynamic control of signal transmission by the ras/erk module. Cell. 2013;155(6):1422–1434. - PMC - PubMed

MeSH terms

Substances