Biology subjects

O'Sullivan, N.

Publications and source records attributed to O'Sullivan, N..

3 recordsLinked to original sources

bioRxiv2026

An organ-resolved rat FFPE phosphoproteome map enables directional kinase activity inference

Formalin-fixed paraffin-embedded (FFPE) tissue is the dominant clinical pathology resource yet whether it faithfully preserves organ signalling biology and supports directional regulatory analysis remains unquantified. We generated a phosphoproteome map from eight healthy rat organs, separating preservation effects from biological variation. Using mass spectrometry, we quantified 54,710 phosphosites on 5,994 proteins across receptors, kinase cascades and nuclear regulators. Organ-specific phosphosite signatures matched known physiological and proliferative states. Paired antagonistic phosphosites converted into "activating-minus-inhibitory" indices that quantified net tissue-specific pathway activity, while a "kinase-by-organ activity" matrix resolved functional hierarchies. Joint analysis with an external fresh-frozen phosphoproteome dataset yielded 58,631 phosphosites total, recovering 86% of the 28,888 sites detected in the frozen dataset. Organ identity explained over 92% of the total variance after batch correction, versus under 0.5% for preservation method. Per-organ phosphosite intensities agreed closely between preservation modes except in brain. This establishes that archived pathology tissue supports biologically faithful phosphoproteome analysis at organ, pathway, and site resolution, providing a framework for retrospective signalling studies in clinical archives. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/741173v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@191c1a2org.highwire.dtl.DTLVardef@3f8d21org.highwire.dtl.DTLVardef@4a8525org.highwire.dtl.DTLVardef@6b4b91_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2026

High-Speed Mass Spectrometers diminish the difference between Data-Dependent and Data-Independent Acquisition Proteomics

Data-dependent acquisition mass spectrometry (DDA-MS) and data-independent acquisition mass spectrometry (DIA-MS) have historically offered complementary strengths in bottom-up proteomics, with DDA providing high-selectivity spectra for post-translational modification (PTM) analysis and DIA enabling more systematic peptide sampling. Here, we asked if this is still the case for the Orbitrap Astral platform that offers high-speed DDA and (ultra-) narrow-window DIA (nDIA) capabilities across proteome and phosphoproteome applications. When DDA and DIA measurements were parameter-matched (to the extent possible), the differences in analytical performance diminished markedly. Across extensive replicate analyses, both methods continued to identify new peptides and proteins without reaching saturation, indicating that the molecular complexity of biological samples still overwhelms even the fastest liquid chromatography-MS (LC-MS) methods. Incomplete sampling also contributed to substantial peptide-level non-overlap between DDA and nDIA and data completeness was only modestly better for nDIA than DDA across many replicates. Quantitatively, DDA and nDIA showed broadly similar precision and accuracy, with nDIA offering slightly higher precision and DDA slightly better accuracy in controlled mixture experiments. MS1-based quantification outperformed MS2-based quantification, particularly for short gradients, supporting MS1 quantification as a robust and general strategy for high-throughput proteomics. In phosphoproteomic samples, DDA and nDIA identified similar numbers of phosphopeptides, but DDA retained a small edge for phosphorylation site localisation. Together, the results show that advances in acquisition speed and sensitivity are narrowing the historical gap between DDA and DIA, while also revealing that current LC-MS workflows remain far from providing comprehensive proteome coverage. Going forward, further gains in dynamic range, scan speed, sensitivity, and transparent software tools will be required to reach systematic, comprehensive and reliable measurements of complex proteomes in a single shot.

biochemistry↗

bioRxiv2025

High-throughput chemical proteomics workflow for profiling protein citrullination dynamics

Citrullination is a post-translational modification implicated in autoimmune and inflammatory diseases, yet its low abundance and lack of effective enrichment tools have limited proteome-wide analysis. Here, we developed a robust chemical proteomics workflow with improved specificity and throughput. This method builds upon glyoxal-based derivatization and incorporates a cleavable biotin linker for efficient peptide enrichment, release, and identification via mass spectrometry. Benchmarking across biological systems demonstrated a >10-fold increase in the detection of citrullinated peptides (> 150-fold increase in intensity) at sub-0.1% abundance. Applying the workflow to mouse brain tissue and human primary neutrophils revealed dynamics and condition-specific changes in the citrullinome, including previously uncharacterized sites and regulatory processes. Notably, extensive citrullination of linker histone H1 and structural proteins such as lamin B1 in ionomycin-activated neutrophils suggests broad remodeling of cell architecture via citrullination. This workflow enables proteome-wide mapping of citrullination sites and facilitates its study across diverse biological contexts.

biochemistry↗