Biology subjects

Jensen, P. T.

Publications and source records attributed to Jensen, P. T..

3 recordsLinked to original sources

bioRxiv2026

STEP-PTMs: Sequential TMT-based Enrichment and Profiling of Post-Translational Modifications

Comprehensive characterization of protein abundance and multiple post-translational modifications (PTMs) from the same biological samples is essential for understanding cellular regulation and PTM crosstalk but remains analytically challenging. Here, we present STEP-PTM (Sequential Tag-based Enrichment of Post-Translational Modifications), a modular TMT-multiplexed workflow that enables integrated quantitative analysis of the proteome, metabolome and multiple PTM classes from a single peptide preparation. Proteins are digested, isobarically labeled using tandem mass tags (TMT), and combined into a single multiplexed peptide pool prior to sequential PTM enrichment, thereby minimizing technical variability, reducing sample requirements and facilitating direct quantitative integration across datasets. STEP-PTM supports flexible sequential enrichment of phosphopeptides, peptides containing free and reversibly modified cysteines, sialylated N-linked glycopeptides, lysine-acetylated peptides and S-palmitoylated peptides, while preserving non-modified peptides for global proteome analysis. PTM-specific database searches further improve identification confidence and quantitative accuracy, and the modular workflow can readily be adapted by incorporating or omitting enrichment modules according to the biological question. Application of STEP-PTM to TMT16-plex cerebral brain organoids enabled the quantification of 10,413 proteins, 2,969 metabolites, 19,655 phosphopeptides, 28,876 peptides containing reversibly modified cysteines, 9,723 peptides containing free cysteines, 1,716 intact sialylated N-linked glycopeptides and 771 lysine-acetylated peptides from the same biological samples. We further demonstrate the applicability of the workflow to multiple mouse tissues, highlighting its broad utility for integrated systems-level characterization of protein expression and PTM regulation across diverse biological models.

biochemistry↗

bioRxiv2026

Modulation of Cysteine modifications upon short time T-cell activation

T cell activation is initiated by rapid and tightly regulated signaling events that rely on reversible post-translational modifications to ensure speed, specificity, and fidelity. While phosphorylation is well established as a central regulator of T cell receptor signaling, the contribution of cysteine-based modifications remains poorly defined at a systems level. Due to their redox-sensitive thiol side chains, cysteine residues can undergo rapid and reversible chemical modifications, enabling them to function as molecular switches that modulate protein structure, activity, and interactions during signal transduction. Engagement of the TCR induces localized redox signaling, primarily through mitochondrial and NADPH oxidase-derived H2O2, leading to selective and reversible oxidation of reactive cysteine residues. These redox events act as spatially and temporally controlled signaling mechanisms rather than global oxidative stress, transiently reshaping phosphorylation-dependent networks through modulation of redox-sensitive kinases, phosphatases, and adaptor proteins. Specificity is conferred by peroxiredoxin-, thioredoxin-, and glutathione-dependent redox control systems that fine-tune cysteine reactivity. In addition, redox-dependent modulation of cysteine residues within zinc-coordinating motifs suggests a coupled redox-zinc signaling axis during T cell activation, where transient redistribution of zinc from cysteine-rich zinc-finger proteins may further regulate kinase activity and transcriptional programs downstream of TCR engagement. Here, we present a quantitative, time-resolved proteomic strategy to systematically interrogate cysteine dynamics during the earliest phases of human T cell activation. By combining cysteine-specific phosphonate adaptable tagging, thiol-disulfide exchange chromatography and TMT-based multiplexed mass spectrometry, we quantified 25,324 free cysteine-containing peptides and 14,853 reversibly modified cysteine peptides across seconds-to-minutes following TCR stimulation. This comprehensive cysteine proteome revealed rapid remodeling of cysteine redox states as early as 30 seconds after activation of T-cells. Functionally, regulated cysteines were enriched in core signaling pathways, including kinases, phosphatases, transcription factors, and nuclear zinc-finger proteins. Notably, key TCR signaling components such as Lck, CD45, ZAP-70, Akt, NFATC3, and RASGRP1 exhibited dynamic cysteine modulation. Together, these findings establish cysteine redox dynamics as a pervasive and functionally relevant regulatory layer in T cell activation, positioning cysteine modification alongside phosphorylation as a central component of immune signal transduction.

systems biology↗

bioRxiv2023

Enrichment of Cysteine S-palmitoylation using Sodium Deoxycholate Acid Precipitation - SDC-ACE

S-palmitoylation is a poorly understood post-translational modification that is gaining more attention as an essential regulator of cellular processes. The reversible nature of S-palmitoylation allows for fine-tuned control of cellular events and adaptation to stimuli. The enrichment of S-palmitoylated proteins and peptides includes the Acyl-Biotin Exchange (ABE) method, Acyl resin-assisted Capture (Acyl-RAC), metabolic labelling, and derivatives thereof. We present a novel method of enrichment of S-palmitoylated peptides termed SDC Acid Precipitation Enrichment (SDC-ACE). Here, S-palmitoylated peptides are enriched by taking advantage of their co-precipitation with Sodium-Deoxycholate (SDC) under acidic conditions, allowing easy and fast separation of lipidated peptides from the sample suspension. We applied our novel method for the characterization of the mouse brain, providing an in-depth analysis of S-palmitoylation events within the brain and comprehensive profile of the mouse brain S-palmitoylome. Furthermore, we applied our method for mapping mouse tissue-specific S-palmitoylation, highlighting the extensive role of S-palmitoylation throughout various organs in the body. Finally, we applied our methods for studying the brain palmitoylome of diabetic db/db mouse, uncovering alterations in the palmitoylation related to obesity and type 2 diabetes. The SDC-ACE method allows fast and easy enrichment of S-palmitoylated peptides, providing a valuable tool for exploring the dynamics and function of S-palmitoylation in diverse biological systems.

biochemistry↗