Biology subjects

Stokes, M. P.

Publications and source records attributed to Stokes, M. P..

5 recordsLinked to original sources

bioRxiv2025

Expanding the Global Map of Protein Post Translational Modifications with Immunoaffinity Enrichment and nDIA Analysis on the Orbitrap Astral Mass Spectrometer

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.

biochemistry↗

bioRxiv2024

Site-specific quantification of the in vivo UFMylome reveals myosin modification in ALS

UFMylation is a ubiquitin-like protein post-translational modification of Ubiquitin Fold Modifier 1 (UFM1) applied to substrate proteins. The UFMylation system is important for normal development and plays a critical role in a variety of cellular processes including regulating telomere length, stress responses and protein quality control. Here, we describe the development of an antibody-based enrichment approach to immunoprecipitate in vivo remnant UFMylated peptides and identification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). We used this approach to identify >200 UFMylation sites from various mouse tissues revealing extensive modification in skeletal muscle. Furthermore, we show that UFMylation is increased in skeletal muscle biopsies from people living with Amyotrophic Lateral Sclerosis (plwALS). Quantification of UFMylation sites in these participant biopsies with multiplexed isotopic labeling and LC-MS/MS reveal prominent increases in myosin UFMylation. Finally, in silico modelling suggest UFMylation of myosin directly adjacent to the ATP-binding site may regulate stability and/or function. Our data suggest that although UFMylation is not as widespread as ubiquitylation, its in vivo status is more complex than previously thought.

biochemistry↗

bioRxiv2023

Identification of chemotherapy targets reveals a nucleus-to-mitochondria ROS sensing pathway

Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, its unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H2O2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H2O2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.

molecular biology↗

bioRxiv2020

Acetylation-mediated phase control of the nucleolus regulates cellular acetyl-CoA responses

The metabolite acetyl-CoA serves as an essential element for a wide range of cellular functions including ATP production, lipid synthesis and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using CRISPR-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa HDAC deacetylases regulating the phase state of the nucleolus. Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

cell biology↗

bioRxiv2019

Deep characterization of the protein lysine acetylation in human gut microbiome and its alterations in patients with Crohn’s disease

Metagenomic and metaproteomic approaches have been used to study the composition and functions of the microbiota. However, no studies have examined post-translational modifications (PTM) on human microbiome proteins at the metaproteome level, and it remains unknown whether the microbial PTM is altered or not in patient microbiome. Herein we used anti-acetyl-lysine (Kac) antibody enrichment strategy and mass spectrometry to characterize the protein lysine acetylation in human microbiome, which successfully identified 35,200 Kac peptides corresponding to 31,821 Kac sites from the microbial or host proteins in human gut microbiome samples. The gut microbial proteins exhibited Kac motifs that were distinct from those of human proteins. Functional analysis showed that microbial Kac proteins were significantly enriched in energy production and abundant in enzymes related to transferases and oxidoreductases. Applying to the analysis of pediatric Crohns disease (CD) patient microbiome identified 52 host and 136 microbial protein Kac sites that were differentially abundant in CD versus controls. Interestingly, most of the decreased Kac sites in CD were derived from Firmicutes and most of the increased sites were derived from Bacteroidetes. Forty-six out of the 52 differentially abundant human protein Kac sites were increased in CD patients, including those on calprotectin, lactotransferrin and immunoglobulins. Taken together, this study provides an efficient approach to study the lysine acetylation in microbiome and revealed taxon-specific alterations in the lysine acetylome as well as changes in host protein acetylation levels in intestinal samples during the on-set of disease in CD patients.

systems biology↗