High-throughput proteomic and phosphoproteomic analysis of formalin-fixed paraffin-embedded tissue

Haines, M.; Thorup, J. R.; Gohsman, S.; Ctortecka, C.; Newton, C.; Rohrer, D. C.; Hostetter, G.; Mani, D. R.; Gillette, M. A.; Satpathy, S.; Carr, S. A.

doi:10.1101/2024.11.17.624038

bioRxiv · 10.1101/2024.11.17.624038

High-throughput proteomic and phosphoproteomic analysis of formalin-fixed paraffin-embedded tissue

Haines, M.·Thorup, J. R.·Gohsman, S.·Ctortecka, C.·Newton, C.·Rohrer, D. C.·Hostetter, G.·Mani, D. R.·Gillette, M. A.·Satpathy, S.·Carr, S. A.

View original source ↗Export metadata ↓

Abstract

Formalin-fixed, paraffin-embedded (FFPE) patient tissues are a valuable resource for proteomic studies with the potential to associate the derived molecular insights with clinical annotations and outcomes. Here we present an optimized, partially automated workflow for FFPE proteomics combining pathology-guided macro-dissection, Adaptive Focused Acoustics (AFA) sonication for lysis and decrosslinking, S-Trap digestion to peptides, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using Orbitrap, Astral or timsTOF HT instrumentation. The workflow enables analysis of up to 96 dissected FFPE tissue samples or 10 {micro}m scrolls, identifying 8,000-10,000 unique proteins per sample with median CVs <20%. Key optimizations include improved tissue lysis strategies, protein quantification for normalization, and peptide cleanup prior to LC-MS/MS analysis. Application to lung adenocarcinoma (LUAD) FFPE blocks confirms the platforms effectiveness in processing complex, clinically relevant samples, achieving deep proteome coverage and quantitative robustness comparable to TMT-based methods. Using the newly released Orbitrap Astral with short, 24-minute gradients, the workflow identifies up to [~]10,000 unique proteins and [~]11,000 localized phosphosites in LUAD FFPE tissue. This high-throughput, scalable workflow advances biomarker discovery and proteomic research in archival tissue samples.

Source connections

Published article ↗

Explore related subjects

systems biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Haines, M., Thorup, J. R., Gohsman, S., Ctortecka, C., Newton, C., Rohrer, D. C., Hostetter, G., Mani, D. R., Gillette, M. A., Satpathy, S., Carr, S. A.. 2024-11-18. High-throughput proteomic and phosphoproteomic analysis of formalin-fixed paraffin-embedded tissue. https://doi.org/10.1101/2024.11.17.624038

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Limit-pushing overexpression reveals constraints on protein abundance

Proteins are often classified as toxic or non-toxic without measuring the abundance reached, leaving constraints on tolerable protein abundance unresolved. We established a limit-pushing approach in Saccharomyces cerevisiae combining strong inducible expression with gTOW-mediated high-copy selection to counteract copy-number compensation while measuring protein abundance and growth. Nearly all of approximately 80 chromosome I proteins severely inhibited growth or reduced viability at sufficiently high abundance. We established IE50, the expression level associated with a 50% reduction in growth rate, to quantify their widely varying overexpression tolerance. IE50 was positively associated with predicted structural order and cytoplasmic localization propensity and negatively associated with sulphur content. Single-cell imaging linked higher tolerance to proteins remaining cytoplasmic without becoming aggregation-positive and revealed abundance-dependent changes in localization and organelle morphology. At extreme abundance, Fun12, Nup60, and Pex22 generated distinct large-scale intracellular states through specific sequence regions. These findings establish overexpression toxicity as a quantitative property linked to protein characteristics and reveal both constraints on tolerable abundance and sequence-dependent capacities for intracellular organization.

systems biology↗

bioRxiv2026

Accessing Enzyme Kinetic Data and Prediction Methods at Scale

Enzyme kinetic parameters inform metabolic models, yet experimental measurements are sparse. A growing body of work predicts them from protein and substrate features, but software fragmentation hinders adoption, so downstream tools lock into the most accessible method. We present OpenKinetics Predictor (at predictor.openkinetics.org), an open-source platform integrating thirteen methods in isolated environments behind one interface. The platform optionally reports similarity between query proteins and each method's training data to contextualise reliability. A common featurisation-prediction abstraction keeps it extensible, and independent parties, including original authors, contributed many methods. We pair it with a data portal (at data.openkinetics.org) that exposes CatLog, a curated kinetic dataset, with precomputed embeddings, predicted binding sites, and standardised splits. Both offer a web interface and an API, and the GECKO modelling toolbox already calls the predictor API. As a case study, we predict across an E. coli model and find inter-predictor agreement varies with metabolic context and data availability.

systems biology↗

bioRxiv2026

A thermoregulatory design principle for transitions into hypometabolism

Mammals entering torpor or hibernation undergo an abrupt transition from normothermia to hypothermia, yet how thermoregulation enables this switch remains poorly understood. Here, we identify dynamical signatures that precede these transitions and a mathematical principle that can generate them. In fasting-induced torpor in mice, body-temperature fluctuations increased before torpor onset, providing an early-warning signal that tracked proximity to the transition better than temperature decline alone. A heat-balance model showed that reducing how strongly the effective heat-loss coefficient depends on body temperature reorganizes thermoregulatory stability, allowing a low-temperature equilibrium to emerge while the normothermic state remains stable. This organization is consistent with a symmetry-broken pitchfork involving a saddle-node. Similar increases in temperature fluctuations preceded hibernation onset in hamsters. These findings link pre-transition temperature dynamics to changes in the underlying thermoregulatory landscape and provide a framework for detecting and understanding transitions from normothermia to hypothermia.

systems biology↗