Abstract
The analytic platform described in this chapter uses proteins extracted from cultured cells as an infinite source of material to set up, validate, and quality control an RPPA platform. Readout of the arrays uses near-infrared fluorescence labeling and data normalization is performed using the bioinformatics package NormaCurve.
In the first part, we will describe the advantages, drawbacks, and different applications of cell line material for RPPA. In the second part, we will describe how the staining protocol, the method of readout, and the normalization method applied afterward are interconnected and should be considered together. Finally, we will describe the NormaCurve package, which is freely available, and its requirements for implementation.
Four protocols are provided in this chapter: (1) Protein lysis of cell lines using a homemade Laemmli buffer, (2) RPPA staining for fluorescent readout including a signal amplification step, (3) total protein staining in the visible spectrum for normalization purposes, and (4) total protein staining in the near-infrared spectrum for normalization purposes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Anderson T, Wulfkuhle J, Liotta L, Winslow RL, Petricoin E 3rd (2009) Improved reproducibility of reverse-phase protein microarrays using array microenvironment normalization. Proteomics 9(24):5562–5566
Aslan O, Cremona M, Morgan C, Cheung LW, Mills GB, Hennessy BT (2018) Preclinical evaluation and reverse phase protein Array-based profiling of PI3K and MEK inhibitors in endometrial carcinoma in vitro. BMC Cancer 18(1):168. https://doi.org/10.1186/s12885-12018-14035-12880
Berlier JE, Rothe A, Buller G, Bradford J, Gray DR, Filanoski BJ, Telford WG, Yue S, Liu J, Cheung CY, Chang W, Hirsch JD, Beechem JM, Haugland RP, Haugland RP (2003) Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates. J Histochem Cytochem 51(12):1699–1712. https://doi.org/10.1177/002215540305101214
Brase JC, Mannsperger H, Sultmann H, Korf U (2011) Antibody-mediated signal amplification for reverse phase protein array-based protein quantification. Methods Mol Biol 785:55–64
Breslin S, O’Driscoll L (2013) Three-dimensional cell culture: the missing link in drug discovery. Drug Discov Today 18(5–6):240–249. https://doi.org/10.1016/j.drudis.2012.1010.1003
Bruna A, Rueda OM, Greenwood W, Batra AS, Callari M, Batra RN, Pogrebniak K, Sandoval J, Cassidy JW, Tufegdzic-Vidakovic A, Sammut SJ, Jones L, Provenzano E, Baird R, Eirew P, Hadfield J, Eldridge M, McLaren-Douglas A, Barthorpe A, Lightfoot H, O’Connor MJ, Gray J, Cortes J, Baselga J, Marangoni E, Welm AL, Aparicio S, Serra V, Garnett MJ, Caldas C (2016) A biobank of breast cancer explants with preserved intra-tumor heterogeneity to screen anticancer compounds. Cell 167(1):260–274.e222. https://doi.org/10.1016/j.cell.2016.1008.1041
Calvert VS, Tang Y, Boveia V, Wulfkuhle J, Schutz-Geschwender A, Olive DM, Liotta LA, Petricoin EF (2004) Development of multiplexed protein profiling and detection using near infrared detection of reverse phase protein microarrays. Clin Proteomics 1(1):81–89
Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, MacLeod RA, Masters JR, Nakamura Y, Reid YA, Reddel RR, Freshney RI (2010) Check your cultures! A list of cross-contaminated or misidentified cell lines. Int J Cancer 127(1):1–8. https://doi.org/10.1002/ijc.25242
Chan SM, Ermann J, Su L, Fathman CG, Utz PJ (2004) Protein microarrays for multiplex analysis of signal transduction pathways. Nat Med 10(12):1390–1396. https://doi.org/10.1038/nm1139
Cree IA (2011) Principles of cancer cell culture. Methods Mol Biol 731:13–26. https://doi.org/10.1007/1978-1001-61779-61080-61775_61772
Creighton CJ (2013) Widespread molecular patterns associated with drug sensitivity in breast cancer cell lines, with implications for human tumors. PLoS One 8(12):e71158. https://doi.org/10.71371/journal.pone.0071158
Dupuy L, Gauthier C, Durand G, Musnier A, Heitzler D, Herledan A, Sakanyan V, Crepieux P, Reiter E (2009) A highly sensitive near-infrared fluorescent detection method to analyze signalling pathways by reverse-phase protein array. Proteomics 9(24):5446–5454
Edmondson R, Broglie JJ, Adcock AF, Yang L (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12(4):207–218. https://doi.org/10.1089/adt.2014.1573
El Botty R, Coussy F, Hatem R, Assayag F, Chateau-Joubert S, Servely JL, Leboucher S, Fouillade C, Vacher S, Ouine B, Cartier A, de Koning L, Cottu P, Bieche I, Marangoni E (2018) Inhibition of mTOR downregulates expression of DNA repair proteins and is highly efficient against BRCA2-mutated breast cancer in combination to PARP inhibition. Oncotarget 9(51):29587–29600. https://doi.org/10.18632/oncotarget.25640
Erdem C, Nagle AM, Casa AJ, Litzenburger BC, Wang YF, Taylor DL, Lee AV, Lezon TR (2016) Proteomic screening and lasso regression reveal differential signaling in insulin and insulin-like Growth Factor I (IGF1) pathways. Mol Cell Proteomics 15(9):3045–3057. https://doi.org/10.1074/mcp.M3115.057729
Espina V, Woodhouse EC, Wulfkuhle J, Asmussen HD, Petricoin EF 3rd, Liotta LA (2004) Protein microarray detection strategies: focus on direct detection technologies. J Immunol Methods 290(1–2):121–133. https://doi.org/10.1016/j.jim.2004.1004.1013
Faget L, Hnasko TS (2015) Tyramide signal amplification for immunofluorescent enhancement. Methods Mol Biol 1318:161–172. https://doi.org/10.1007/1978-1001-4939-2742-1005_1016
Gundisch S, Grundner-Culemann K, Wolff C, Schott C, Reischauer B, Machatti M, Groelz D, Schaab C, Tebbe A, Becker KF (2013) Delayed times to tissue fixation result in unpredictable global phosphoproteome changes. J Proteome Res 12(10):4424–4434. https://doi.org/10.1021/pr400451z
Hu J, He X, Baggerly KA, Coombes KR, Hennessy BT, Mills GB (2007) Non-parametric quantification of protein lysate arrays. Bioinformatics 23(15):1986–1994
Jiang R, Mircean C, Shmulevich I, Cogdell D, Jia Y, Tabus I, Aldape K, Sawaya R, Bruner JM, Fuller GN, Zhang W (2006) Pathway alterations during glioma progression revealed by reverse phase protein lysate arrays. Proteomics 6(10):2964–2971. https://doi.org/10.1002/pmic.200500555
Ju Z, Liu W, Roebuck PL, Siwak DR, Zhang N, Lu Y, Davies MA, Akbani R, Weinstein JN, Mills GB, Coombes KR (2015) Development of a robust classifier for quality control of reverse-phase protein arrays. Bioinformatics 31(6):912–918. https://doi.org/10.1093/bioinformatics/btu1736
Kaushik P, Molinelli EJ, Miller ML, Wang W, Korkut A, Liu W, Ju Z, Lu Y, Mills G, Sander C (2014) Spatial normalization of reverse phase protein array data. PLoS One 9(12):e97213. https://doi.org/10.91371/journal.pone.0097213
Koplev S, Lin K, Dohlman AB, Ma’ayan A (2018) Integration of pan-cancer transcriptomics with RPPA proteomics reveals mechanisms of epithelial-mesenchymal transition. PLoS Comput Biol 14(1):e1005911. https://doi.org/10.1371/journal.pcbi.1005911
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Liu W, Ju Z, Lu Y, Mills GB, Akbani R (2014) A comprehensive comparison of normalization methods for loading control and variance stabilization of reverse-phase protein array data. Cancer Inform 13:109–117. https://doi.org/10.4137/CIN.S13329
Loebke C, Sueltmann H, Schmidt C, Henjes F, Wiemann S, Poustka A, Korf U (2007) Infrared-based protein detection arrays for quantitative proteomics. Proteomics 7(4):558–564
Mannsperger HA, Gade S, Henjes F, Beissbarth T, Korf U (2010a) RPPanalyzer: analysis of reverse-phase protein array data. Bioinformatics 26(17):2202–2203
Mannsperger HA, Uhlmann S, Schmidt C, Wiemann S, Sahin O, Korf U (2010b) RNAi-based validation of antibodies for reverse phase protein arrays. Proteome Sci 8:69
Masters JR (2000) Human cancer cell lines: fact and fantasy. Nat Rev Mol Cell Biol 1(3):233–236. https://doi.org/10.1038/35043102
Mauland KK, Ju Z, Tangen IL, Berg A, Kalland KH, Oyan AM, Bjorge L, Westin SN, Krakstad C, Trovik J, Mills GB, Hoivik EA, Johanna Werner HM (2017) Proteomic profiling of endometrioid endometrial cancer reveals differential expression of hormone receptors and MAPK signaling proteins in obese versus non-obese patients. Oncotarget 8(63):106989–107001. https://doi.org/10.18632/oncotarget.22203
Mendes KN, Nicorici D, Cogdell D, Tabus I, Yli-Harja O, Guerra R, Hamilton SR, Zhang W (2007) Analysis of signaling pathways in 90 cancer cell lines by protein lysate array. J Proteome Res 6(7):2753–2767. https://doi.org/10.1021/pr070184h
Michaut M, Chin SF, Majewski I, Severson TM, Bismeijer T, de Koning L, Peeters JK, Schouten PC, Rueda OM, Bosma AJ, Tarrant F, Fan Y, He B, Xue Z, Mittempergher L, Kluin RJ, Heijmans J, Snel M, Pereira B, Schlicker A, Provenzano E, Ali HR, Gaber A, O'Hurley G, Lehn S, Muris JJ, Wesseling J, Kay E, Sammut SJ, Bardwell HA, Barbet AS, Bard F, Lecerf C, O'Connor DP, Vis DJ, Benes CH, McDermott U, Garnett MJ, Simon IM, Jirstrom K, Dubois T, Linn SC, Gallagher WM, Wessels LF, Caldas C, Bernards R (2016) Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer. Sci Rep 6:18517
Neeley ES, Kornblau SM, Coombes KR, Baggerly KA (2009) Variable slope normalization of reverse phase protein arrays. Bioinformatics 25(11):1384–1389
Neeley ES, Baggerly KA, Kornblau SM (2012) Surface adjustment of reverse phase protein arrays using positive control spots. Cancer Inform 11:77–86
Ngoka LC (2008) Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci 6:30. https://doi.org/10.1186/1477-5956-1186-1130
Nikfarjam L, Farzaneh P (2012) Prevention and detection of mycoplasma contamination in cell culture. Cell J 13(4):203–212
Niu N, Wang L (2015) In vitro human cell line models to predict clinical response to anticancer drugs. Pharmacogenomics 16(3):273–285. https://doi.org/10.2217/pgs.2214.2170
Park ES, Rabinovsky R, Carey M, Hennessy BT, Agarwal R, Liu W, Ju Z, Deng W, Lu Y, Woo HG, Kim SB, Cheong JH, Garraway LA, Weinstein JN, Mills GB, Lee JS, Davies MA (2010) Integrative analysis of proteomic signatures, mutations, and drug responsiveness in the NCI 60 cancer cell line set. Mol Cancer Ther 9(2):257–267. https://doi.org/10.1158/1535-7163.mct-09-0743
Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, Gillespie JW, Emmert-Buck MR, Roth MJ, Petricoin IE, Liotta LA (2001) Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20(16):1981–1989
Rottem S, Naot Y (1998) Subversion and exploitation of host cells by mycoplasmas. Trends Microbiol 6(11):436–440
Troncale S, Barbet A, Coulibaly L, Henry E, He B, Barillot E, Dubois T, Hupe P, de Koning L (2012) NormaCurve: a SuperCurve-based method that simultaneously quantifies and normalizes reverse phase protein array data. PLoS One 7(6):e38686
Varghese RS, Zuo Y, Zhao Y, Zhang YW, Jablonski SA, Pierobon M, Petricoin EF, Ressom HW, Weiner LM (2017) Protein network construction using reverse phase protein array data. Methods 124:89–99. https://doi.org/10.1016/j.ymeth.2017.1006.1017
von der Heyde S, Sonntag J, Kaschek D, Bender C, Bues J, Wachter A, Timmer J, Korf U, Beissbarth T (2014) RPPanalyzer toolbox: an improved R package for analysis of reverse phase protein array data. Biotechniques 57(3):125–135. https://doi.org/10.2144/000114205
Zhang L, Wei Q, Mao L, Liu W, Mills GB, Coombes K (2009) Serial dilution curve: a new method for analysis of reverse phase protein array data. Bioinformatics 25(5):650–654
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Nature Singapore Pte Ltd.
About this chapter
Cite this chapter
Ouine, B., Rajkumar, S., Hupé, P., de Koning, L. (2019). Analytical Platforms 1: Use of Cultured Cells and Fluorescent Read-Out Coupled to NormaCurve Normalization in RPPA. In: Yamada, T., Nishizuka, S., Mills, G., Liotta, L. (eds) Reverse Phase Protein Arrays. Advances in Experimental Medicine and Biology, vol 1188. Springer, Singapore. https://doi.org/10.1007/978-981-32-9755-5_5
Download citation
DOI: https://doi.org/10.1007/978-981-32-9755-5_5
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-32-9754-8
Online ISBN: 978-981-32-9755-5
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)