The requirement of vitamin A for the synthesis of the visual chromophore and the light-sensing pigments has been studied in vertebrate and invertebrate model organisms. To identify the molecular mechanisms that orchestrate the ocular response to vitamin A deprivation, we took advantage of the fact that Drosophila melanogaster predominantly requires vitamin A for vision, but not for development or survival. We analyzed the impacts of vitamin A deficiency on the morphology, the lipidome, and the proteome of the Drosophila eye. We found that chronic vitamin A deprivation damaged the light-sensing compartments and caused a dramatic loss of visual pigments, but also decreased the molar abundance of most phototransduction proteins that amplify and transduce the visual signal. Unexpectedly, vitamin A deficiency also decreased the abundances of specific subunits of mitochondrial TCA cycle and respiratory chain components but increased the levels of cuticle- and lens-related proteins. In contrast, we found no apparent effects of vitamin A deficiency on the ocular lipidome. In summary, chronic vitamin A deficiency decreases the levels of most components of the visual signaling pathway, but also affects molecular pathways that are not vision-specific and whose mechanistic connection to vitamin A remains to be elucidated.

Post-translational modifications (PTMs) contribute greatly to the diversity of the human proteome by affecting protein structure, function, interactions, stability, localization, and more. The study of PTMs is essential to understand various cellular functions, disease mechanisms, and aid in the development of biomarkers and design of therapeutic targets. Owing to their diversity, dynamic nature, and low stoichiometry compared to unmodified proteome counterparts, the analysis of PTMs remains challenging. In this study, immunoaffinity enrichment of PTM peptides was combined with analysis using Data Dependent Acquisition (DDA) and narrow window Data Independent Acquisition (nDIA) on the Orbitrap Astral mass spectrometer (MS) as well as comparative analysis using the Orbitrap Fusion Lumos MS for Ubiquitination, Phosphorylation, Acetylation, Succinylation, and Methylation. Human cell line and mouse tissue samples at various input peptide amounts were immuno-enriched and mass spectrometry data was acquired on both instruments to assess depth of coverage and number of novel sites identified. The study identified a total of 106,152 unique ubiquitin sites, 64,397 phosphorylation sites (43,721 phosphoserine, 8,414 phosphothreonine and 12,262 phosphotyrosine), 14,245 acetylation, 5,272 succinylation and 1,461 mono-methylation sites. In half the acquisition time, nDIA analysis of immuno-enriched samples on Orbitrap Astral MS provided much greater depth of coverage for all PTMs compared to DDA analysis on Orbitrap Fusion Lumos MS, with up to 33-fold more PTM peptides identified and quantified. Overall, the data presented in this study demonstrates the need for enrichment for PTM detection and the utility of combining antibody-based peptide capture and nDIA on the Orbitrap Astral MS as powerful tools for discovery and profiling of protein post-translational modifications in cells and tissues.

Post-translational modifications (PTMs) contribute greatly to the diversity of the human proteome by affecting protein structure, function, interactions, stability, localization, and more. The study of PTMs is essential to understand various cellular functions, disease mechanisms, and aid in the development of biomarkers and design of therapeutic targets. Owing to their diversity, dynamic nature, and low stoichiometry compared to unmodified proteome counterparts, the analysis of PTMs remains challenging. In this study, immunoaffinity enrichment of PTM peptides was combined with analysis using Data Dependent Acquisition (DDA) and narrow window Data Independent Acquisition (nDIA) on the Orbitrap Astral mass spectrometer (MS) as well as comparative analysis using the Orbitrap Fusion Lumos MS for Ubiquitination, Phosphorylation, Acetylation, Succinylation, and Methylation. Human cell line and mouse tissue samples at various input peptide amounts were immuno-enriched and mass spectrometry data was acquired on both instruments to assess depth of coverage and number of novel sites identified. The study identified a total of 88,731 unique ubiquitin sites, 64,397 phosphorylation sites (43,721 phosphoserine, 8,414 phosphothreonine and 12,262 phosphotyrosine), 11,629 acetylation, 5,272 succinylation and 1,461 mono-methylation sites. In half the acquisition time, nDIA analysis of immuno-enriched samples on Orbitrap Astral MS provided much greater depth of coverage for all PTMs compared to DDA analysis on Orbitrap Fusion Lumos MS, with up to 33-fold more PTM peptides identified and quantified. Overall, the data presented in this study demonstrates the need for enrichment for PTM detection and the utility of combining antibody-based peptide capture and nDIA on the Orbitrap Astral MS as powerful tools for discovery and profiling of protein post-translational modifications in cells and tissues.

RNA Polymerase II subunit RPB1 in eukaryotes contains a conserved, repetitive C‐terminal Domain (CTD) consisting of predominantly Tyr ₁ Ser ₂ Pro ₃ Thr ₄ Ser ₅ Pro ₆ Ser ₇ heptad repeats. While Ser ₂ and Ser ₅ are strongly conserved and serve as essential phosphorylation sites for recruiting specific transcription factors, the role of Ser ₇ is less understood as it is not well conserved nor seemingly required for general transcription. However, Ser ₇ is indeed phosphorylated across most genes in vivo suggesting that it is an important mark in transcription. We hypothesized that different mutations and modifications of the Ser ₇ site regulate the phosphorylation of other CTD residues which alter transcriptional outcomes. To test this hypothesis, we first generated GST‐CTD substrates with Ser ₇ mutated to residues with different charges like Glu and Lys to mimic specific chemical modifications or naturally occurring mutations. We then treated these substrates with different CTD kinases and used ultraviolet photodissociation mass spectrometry (UVPD‐MS) to analyze the resulting phosphorylation patterns. We found that the CTD kinase ABL1 preferentially targets Tyr ₁ adjacent to Glu ₇ while phosphorylating Tyr ₁ away from Lys ₇ or Arg ₇ , showing that charge in the seventh CTD position can regulate CTD kinase activity. Furthermore, we observed increased ABL1 phosphorylation of Tyr ₁ adjacent to Gln ₇ which mimics Lys acetylation, suggesting that this CTD kinase regulation is reversible. To test how Ser ₇ modification can affect CTD phosphorylation and transcription in vivo , we used UVPD‐MS and ChIP‐Seq to analyze samples from cells expressing RPB1 with Ser ₇ mutated to Glu in every other repeat. Our analysis provides some of the first in vitro and in vivo evidence that Ser ₇ modification or mutation can control CTD phosphorylation patterns thereby regulating transcriptional outcome. Support or Funding Information NIH 1R01GM125882‐01A1 The Welch Foundation This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .