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

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

doi:10.1016/j.jprot.2022.104596

Cited by 16 publications

(7 citation statements)

References 77 publications

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“…Although RPPA is commonly used in cancer research, its advantages of detecting several hundred proteins in a cost‐effective, sensitive, and high‐throughput manner expand its usage. For example, the RPPA platform has the potential for translational applications by discovering and validating epigenetic states as therapeutic targets and biomarkers 68 . Here, we used RPPA technology to predict PD biomarkers for the first time.…”

Section: Discussionmentioning

confidence: 99%

ErbB2_pY_‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

et al. 2023

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AimsThis study aims to reveal a promising biomarker for Parkinson's disease (PD) based on research with reverse phase protein array (RPPA) technology for the first time and in vivo verification, which gains time for early intervention in PD, thus increasing the effectiveness of treatment and reducing disease morbidity.Methods and ResultsWe employed RPPA technology which can assess both total and post‐translationally modified proteins to identify biomarker candidates of PD in a cellular PD model. As a result, the phosphorylation (pY‐1248) of the epidermal growth factor receptor (EGFR) ErbB2 is a promising biomarker candidate for PD. In addition, lapatinib, an ErbB2 tyrosine kinase inhibitor, was used to verify this PD biomarker candidate in vivo. We found that lapatinib‐attenuated dopaminergic neuron loss and PD‐like behavior in the zebrafish PD model. Accordingly, the expression of ErbB2pY‐1248 significantly increased in the MPTP‐induced mouse PD model. Our results suggest that ErbB2pY‐1248 is a predictive biomarker for PD.ConclusionsIn this study, we found that ErbB2pY‐1248 is a predictive biomarker of PD by using RPPA technology and in vivo verification. It offers a new perspective on PD diagnosing and treatment, which will be essential in identifying individuals at risk of PD. In addition, this study provides new ideas for digging into biomarkers of other neurodegenerative diseases.

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Section: Discussionmentioning

confidence: 99%

ErbB2_pY_‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

et al. 2023

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“…PTMs considered for histone H3.2 include variable K4me1/2/3, K9ac, K9me1/2/3, K14ac, K18ac, K23ac, K27ac, K27me1/2/3, and K36me1/2/3. This data analysis approach has been used extensively in other studies in our lab to reveal quantitative changes in histone proteoforms (Holt et al, 2019;Jiang et al, 2018Jiang et al, , 2019Taylor and Young, 2023;Wang et al, 2018aWang et al, , 2018bWang et al, , 2022. This approach has also been validated against reverse phase protein array based quantitation (Wang et al, 2022).…”

Section: Mass Spectrometry Data Analysismentioning

confidence: 99%

“…This data analysis approach has been used extensively in other studies in our lab to reveal quantitative changes in histone proteoforms (Holt et al, 2019;Jiang et al, 2018Jiang et al, , 2019Taylor and Young, 2023;Wang et al, 2018aWang et al, , 2018bWang et al, , 2022. This approach has also been validated against reverse phase protein array based quantitation (Wang et al, 2022). For analyses of BAT versus liver histone modifications, we conducted multiple unpaired t-tests using the false discovery rate approach, and discoveries were determined using the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli.…”

Section: Mass Spectrometry Data Analysismentioning

confidence: 99%

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress

Taylor,

Steinthal,

Dias

et al. 2023

Preprint

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Regulation of the thermogenic response by brown adipose tissue (BAT) is an important component of energy homeostasis with implications for the treatment of obesity and diabetes. Motivated to understand how BAT function is regulated epigenetically, we reanalyzed publicly available RNA-seq data and uncovered many nodes representing epigenetic modifiers that are altered in BAT in response to chronic thermogenic activation. Thus, we hypothesized that chronic thermogenic activation broadly alters epigenetic modifications of DNA and histones in BAT. To test our hypothesis, wildtype male C57BL/6J mice were housed under chronic conditions of thermoneutral temperature (TN, 28.8°C), mild cold/room temperature (RT, 22°C), or severe cold (SC, 8°C). BAT and reference tissue liver were subsequently dissected from each mouse. Reduced representation bisulfite sequencing (RRBS) reveals decreased methylation of promoters and intragenic regions in BAT genomic DNA in response to varying degrees of chronic cold exposure. Integration of our RRBS and the RNA-Seq dataset suggests a role for epigenetic modification of DNA in gene regulation in response to cold. To analyze histone modifications in BAT, we develop a robust method for the isolation of histones and report the first quantitation of histone H3.2 and H4 proteoforms. We initially observe that BAT and liver exhibit different histone proteoforms. Next, we report housing temperature-dependent changes in histone proteoforms in BAT. We observe differences in bivalent proteoforms such as H3{K9me2,K23ac,K36me1}, H3{K9me3,K23ac,K36me1} and H4<K8acK16acK20me3>. Taken together, our results provide novel findings supporting global epigenetic modification in murine BAT in response to varying degrees of chronic cold stimuli and establish a methodology to quantitatively study histones in BAT, allowing for direct comparisons to decipher mechanistic changes during the thermogenic response.

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“…The RPPA can be used to identify a specific protein expression in all the lysates that are printed on the array . The RPPA has been used to profile the histone modifications in the nuclear extracts or cell lysates. , Since the cell lysates on the RPPA array can serve as the substrates, we propose that the overexpression of a kinase may increase the phosphorylation of the substrates and thus can be screened for kinases. In our study, we constructed RPPA with lysates of 4126 different overexpressed strains from the ASKA library and named E.…”

Section: Introductionmentioning

confidence: 99%

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase

Keskin,

Chen,

Tsai

et al. 2024

Anal. Chem.

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Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.

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

Cited by 16 publications

References 77 publications

ErbB2_pY_‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

ErbB2_pY_‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase

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

Cited by 16 publications

References 77 publications

ErbB2pY‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

ErbB2pY‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

Histone proteoform analysis reveals epigenetic changes in adult mouse brown adipose tissue in response to cold stress

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase

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ErbB2_pY_‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification

ErbB2_pY_‐1248 as a predictive biomarker for Parkinson's disease based on research with RPPA technology and in vivo verification