High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array

Xuan Wang; Zhongcheng Shi; Hsin Yi Lu; Jean J. Kim; Wen Bu; Jose A. Villalobos; Dimuthu N. Perera; Sung Yun Jung; Tao Wang; Sandra L. Grimm; Bethany C. Taylor; Kimal Rajapakshe; Hyekyung Park; Julia Wulfkuhle; Nicolas L. Young; Yi Li; Cristian Coarfa; Dean P. Edwards; Shixia Huang

doi:10.1016/j.jprot.2022.104596

High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array

Xuan Wang, Zhongcheng Shi, Hsin Yi Lu, Jean J. Kim, Wen Bu, Jose A. Villalobos, Dimuthu N. Perera, Sung Yun Jung, Tao Wang, Sandra L. Grimm, Bethany C. Taylor, Kimal Rajapakshe, Hyekyung Park, Julia Wulfkuhle, Nicolas L. Young, Yi Li, Cristian Coarfa, Dean P. Edwards, Shixia Huang

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. Significance: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

Original language	English (US)
Article number	104596
Journal	Journal of Proteomics
Volume	262
DOIs	https://doi.org/10.1016/j.jprot.2022.104596
State	Published - Jun 30 2022
Externally published	Yes

Keywords

Breast cancer
Epigenetics
High-throughput
Induced pluripotent stem cells
Post-translational modifications
RPPA

ASJC Scopus subject areas

Biophysics
Biochemistry

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10.1016/j.jprot.2022.104596

Cite this

Wang, X., Shi, Z., Lu, H. Y., Kim, J. J., Bu, W., Villalobos, J. A., Perera, D. N., Jung, S. Y., Wang, T., Grimm, S. L., Taylor, B. C., Rajapakshe, K., Park, H., Wulfkuhle, J., Young, N. L., Li, Y., Coarfa, C., Edwards, D. P., & Huang, S. (2022). High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array. Journal of Proteomics, 262, Article 104596. https://doi.org/10.1016/j.jprot.2022.104596

Wang, X, Shi, Z, Lu, HY, Kim, JJ, Bu, W, Villalobos, JA, Perera, DN, Jung, SY, Wang, T, Grimm, SL, Taylor, BC, Rajapakshe, K, Park, H, Wulfkuhle, J, Young, NL, Li, Y, Coarfa, C, Edwards, DP & Huang, S 2022, 'High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array', Journal of Proteomics, vol. 262, 104596. https://doi.org/10.1016/j.jprot.2022.104596

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title = "High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array",

abstract = "Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. Significance: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.",

keywords = "Breast cancer, Epigenetics, High-throughput, Induced pluripotent stem cells, Post-translational modifications, RPPA",

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year = "2022",

month = jun,

day = "30",

doi = "10.1016/j.jprot.2022.104596",

language = "English (US)",

volume = "262",

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T1 - High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array

AU - Wang, Xuan

AU - Shi, Zhongcheng

AU - Lu, Hsin Yi

AU - Kim, Jean J.

AU - Bu, Wen

AU - Villalobos, Jose A.

AU - Perera, Dimuthu N.

AU - Jung, Sung Yun

AU - Wang, Tao

AU - Grimm, Sandra L.

AU - Taylor, Bethany C.

AU - Rajapakshe, Kimal

AU - Park, Hyekyung

AU - Wulfkuhle, Julia

AU - Young, Nicolas L.

AU - Li, Yi

AU - Coarfa, Cristian

AU - Edwards, Dean P.

AU - Huang, Shixia

PY - 2022/6/30

Y1 - 2022/6/30

N2 - Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. Significance: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

AB - Epigenetic variation plays a significant role in normal development and human diseases including cancer, in part through post-translational modifications (PTMs) of histones. Identification and profiling of changes in histone PTMs, and in proteins regulating PTMs, are crucial to understanding diseases, and for discovery of epigenetic therapeutic agents. In this study, we have adapted and validated an antibody-based reverse phase protein array (RPPA) platform for profiling 20 histone PTMs and expression of 40 proteins that modify histones and other epigenomic regulators. The specificity of the RPPA assay for histone PTMs was validated with synthetic peptides corresponding to histone PTMs and by detection of histone PTM changes in response to inhibitors of histone modifier proteins in cell cultures. The useful application of the RPPA platform was demonstrated with two models: induction of pluripotent stem cells and a mouse mammary tumor progression model. Described here is a robust platform that includes a rapid microscale method for histone isolation and partially automated workflows for analysis of histone PTMs and histone modifiers that can be performed in a high-throughput manner with hundreds of samples. This RPPA platform has potential for translational applications through the discovery and validation of epigenetic states as therapeutic targets and biomarkers. Significance: Our study has established an antibody-based reverse phase protein array platform for global profiling of a wide range of post-translational modifications of histones and histone modifier proteins. The high-throughput platform provides comprehensive analyses of epigenetics for biological research and disease studies and may serve as screening assay for diagnostic purpose or therapy development.

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KW - Epigenetics

KW - High-throughput

KW - Induced pluripotent stem cells

KW - Post-translational modifications

KW - RPPA

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SN - 1874-3919

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JF - Journal of Proteomics

M1 - 104596

ER -

High-throughput profiling of histone post-translational modifications and chromatin modifying proteins by reverse phase protein array

Abstract

Keywords

ASJC Scopus subject areas

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