Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications.

Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to a perturbation, protein structure may be altered by various molecular events, such as post-translational modifications, protein-protein interactions, aggregation, allostery or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of biological processes and pathways. Here, we present an updated protocol for LiP-MS, a proteomics technique combining limited proteolysis with mass spectrometry, to detect protein structural alterations in complex backgrounds and on a proteome-wide scale. In LiP-MS, proteins undergo a brief proteolysis in native conditions followed by complete digestion in denaturing conditions, to generate structurally informative proteolytic fragments that are analyzed by mass spectrometry. We describe advances in the throughput and robustness of the LiP-MS workflow and implementation of data-independent acquisition-based mass spectrometry, which together achieve high reproducibility and sensitivity, even on large sample sizes. We introduce MSstatsLiP, an R package dedicated to the analysis of LiP-MS data for the identification of structurally altered peptides and differentially abundant proteins. The experimental procedures take 3 d, mass spectrometric measurement time and data processing depend on sample number and statistical analysis typically requires ~1 d. These improvements expand the adaptability of LiP-MS and enable wide use in functional proteomics and translational applications.

Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding.

The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.