Biology subjects

Turner, N. P.

Publications and source records attributed to Turner, N. P..

3 recordsLinked to original sources

bioRxiv2026

Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease, yet mass spectrometry-based histone PTM analysis remains constrained by inefficient sample preparation workflows. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation to hours while improving PTM coverage and quantitative accuracy. Systematic evaluation of Arg-C Ultra and a recombinant (r)-Chymotrypsin protease under varied conditions, including standard derivatization with propionic anhydride and tandem mass tag (TMT) labeling, demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM that exceeds Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMTs tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 58 succinylation and 31 glutarylation sites - a dark epigenome largely undetected by propionylation-based methods. Complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. In HEK293T cells treated with the pan-sirtuin inhibitor nicotinamide, RIPUP quantified 112 statistically significant peptidoforms (adj p < 0.05), predominantly increasing with NAM dose (88 up, 24 down). Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identified >200 PTMs including H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A K118/K119 ubiquitination. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.

biochemistry↗

bioRxiv2025

The effects of Alcohol Dependence on the CSF Proteome in Mice: Evidence for Blood-Brain Barrier Dysfunction and Neuroinflammation

Alcohol use disorder (AUD) represents a significant neurological health burden, yet the biological mechanisms underlying alcohol-induced brain pathology remain incompletely understood. Moreover, the molecular underpinnings of the transition from alcohol exposure to alcohol dependence are not well-characterized. We used mass spectrometry (MS)-based proteomics in a preliminary discovery study to compare cerebrospinal fluid (CSF) of alcohol-exposed Non-dependent (Non-dep) versus alcohol-dependent (Dep) mice that underwent the chronic intermittent ethanol (alcohol) - two-bottle choice (CIE-2BC) procedure and systemic anti-IL-6 Receptor antibody administration. CSF samples from individual mice were processed for proteomic analysis and digested with trypsin overnight. Peptides were analyzed via data-independent acquisition (DIA)-MS and data were processed in DIA-NN at 1% FDR. We identified 611 unique proteins across both groups, with 140 proteins differentially detected in CSF from Dep mice and 67 proteins specific to alcohol-exposed but Non-dep controls. The Dep-specific proteins revealed signatures of blood-brain barrier (BBB) disruption, neuroinflammation, cellular stress responses, and complement system activation. In contrast, Non-dep-specific proteins indicated preserved protective mechanisms including complement regulation, anti-inflammatory signaling, and neuronal calcium homeostasis. Ethanol-dependent-specific findings include MMP2, BIP, and to a lesser extent VE-cadherin (CDH5) and VCAM1, indicative of the beginnings of endothelial damage and BBB disruption, alongside established neuroinflammation markers GFAP, CHI3L1, and CX3CL1. This work provides novel preliminary protein-level evidence that alcohol exposure and alcohol dependence are dichotomous; despite the small sample size and limited power for moderate effect sizes, there appears to be a clear molecular transition from maintained protective mechanisms to vascular damage, BBB breakdown, and sustained neuroinflammation.

neuroscience↗

bioRxiv2023

Cross-species proteomic and microRNA comparison of extracellular vesicles in human milk, cows milk, and infant formula products: moving towards next generation infant formula products.

Milk and milk products such as infant formula (IF) play a fundamental role in serving the nutritional needs of the developing infant. Extracellular vesicles (EVs) in human (HM) and cows milk (CM) contain molecular cargo such as proteins and micro(mi)RNA that serve as functional messengers between cells and may be of importance to infant health. Here, we have developed a pipeline using advanced proteomics and transcriptomics to enable cross-species comparison of milk and IF EVs. EVs from HM, CM and IF were subjected to data-independent acquisition mass spectrometry and RNA-seq. Differentially abundant proteins (143) and miRNAs (514) were identified in HM and CM EVs, and CM EV proteins and miRNAs were preserved in IF EVs ([~]20% protein; [~]90% miRNA). We foresee this work to be used in large scale studies to determine biologically relevant species-specific differences in milk EVs that could be leveraged to improve IF products.

biochemistry↗