Phosphoproteomic Analysis Of Thermophilic Prokaryotes: Expanding The Phosphorylation Landscape In Extreme Environments

Mansi Jain1, Ana Carvalho2,4 , Julian Quehenberger2, Josephine Boel Andresen3, Eveline Peeters3, Eva Csosz1 INTRODUCTION RESULTS Phosphoproteomic Analysis Of Thermophilic Prokaryotes: Expanding The Phosphorylation Landscape In Extreme Environments 1Proteomics Core Facility, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary 2NovoArc GmbH, Austria 3 Microbiology Research Group, Department of Bioengineering sciences, Vrije Universiteit Brussel , Belgium 4 Department of Analytical Chemistry, Doctoral School in Chemistry, University of Vienna, Austria CONCLUSION Protein phosphorylation is a key post-translational modification that regulates numerous cellular processes, including signal transduction, enzyme activity, and stress responses. In thermophilic organisms, which thrive under extreme heat conditions, phosphorylation may play an essential role in maintaining cellular stability and enabling adaptation to thermal stress. This study investigates sample preparation methods for proteomic analysis of thermophilic organisms, which possess heat-resistant cellular structures. We compared various lysis and digestion protocols on species such as Sulfolobus acidocaldarius, Haloferax volcanii, Caldimonas thermodepolymerans, and Parageobacillus thermoglucosidasius to optimize protein extraction from these resilient cells. Using advanced techniques like LC-ESI-MS/MS, we evaluated digestion efficiency and assessed different software tools and protein identification criteria, including the use of proteotypic peptides to establish an optimal workflow for subsequent Phosphopeptides enrichment. These efforts aim to deepen our understanding of regulatory mechanisms and adaptation strategies in organisms living under extreme thermal conditions. . In comparison to total-cell lysate samples, which highlight a broad array of catalytic, binding, and metabolic functions, phosphoproteins in Haloferax volcanii are distinctly enriched for ATP-dependent chaperoning and signaling roles, whereas in Sulfolobus acidocaldarius they primarily influence ribosome structure and oxidoreductase activities. This underscores that phosphorylation selectively modulates proteins crucial for cellular regulation and adaptation, in contrast to the general proteome, where highly abundant proteins predominate. To address this bias and gain deeper insights into phosphorylation events, we simultaneously optimized our experimental workflow to enrich phosphoproteins, enabling a more comprehensive and accurate characterization of regulatory phosphorylation in thermophilic proteomes. Among tested methods, 100% TFA cell lysis with insolution digestion using a trypsin/Lys-C enzyme cocktail provided the best overall performance, being simple, fast, and cost-effective, requiring no specialized equipment and integrating seamlessly with DIA-NN analysis. By taking proteotypicity into account for protein group filtration rather than relying solely on two-peptide hits, this approach further improved identification accuracy. ACKNOWLEDGEMENTS METHODOLOGY 0 500 1000 1500 2000 Haloferax volcanii Caldimonas thermodepolymerans Parageobacilus thermoglucosidasius Sulfolobus acidocaldarius Trypsin/LysC Trypsin Protease Performance Comparison Trypsin vs. Trypsin/LysC Sample Preparation Methods Y-axis Labels (A–D) A —Detergent lysis ,On column B —Detergent lysis, In solution C —Acid Lysis, On Column D —Acid Lysis, In Solution Haloferax volcanii Caldimonas thermodepolymerans Parageobacilus thermoglucosidasius Sulfolobus acidocaldarius 0 500 1000 1500 D C B A 0 500 1000 1500 D C B A 0 500 1000 1500 D C B A 0 200 400 600 800 D C B A Cell Pellet Proteins Peptides Phosphopeptides LC-MS/MS Analysis Software Analysis Protein group filtering Functional Enrichment Analysis Protein Extraction Protein Digestion Acid Lysis In-Solution Digestion Trypsin/LysC ReSynBio Ti-IMAC HP beads Phosphoproteins Identified in Total Cell Lysate 0 10 20 30 40 50 Haloferax volcanii Caldimonas thermodepolymerans Parageobacilus thermoglucosidasius Sulfolobus acidocaldarius This research was funded by 2020-2.1.1-ED-2023-00269 and cofounded by 101119980 —PROHITS —HORIZON-MSCA2022-DN-01. Comparison of Different Sample Preparation Methods Protein Groups Protein Groups Sulfolobus acidocaldarius Haloferax volcanii Workflows Tested for Sample Preparation Optimized Sample Preparation Workflow Total Cell Lysate Phosphoproteins Identified In Cell Lysate Functional Enrichment Analysis Based on Gene Ontology Protein Groups Protein GroupsProtein Groups Protein Groups