bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2021.04.14.439901

Profiling the Human Phosphoproteome to Estimate the True Extent of Protein Phosphorylation

Kalyuzhnyy, A.·Eyers, P. A.·Eyers, C. E.·Sun, Z.·Deutsch, E. W.·Jones, A. R.
View original source ↗Export metadata ↓

Abstract

Mass spectrometry-based phosphoproteomics allows large-scale generation of phosphorylation site data. However, analytical pipelines need to be carefully designed and optimised to minimise incorrect identification of phosphopeptide sequences or wrong localisation of phosphorylation sites within those peptides. Public databases such as PhosphoSitePlus (PSP) and PeptideAtlas (PA) compile results from published papers or openly available MS data, but to our knowledge, there is no database-level control for false discovery of sites, subsequently leading to the likely overestimation of true phosphosites. It is therefore difficult for researchers to assess which phosphosites are "real" and which are likely to be artefacts of data processing. By profiling the human phosphoproteome, we aimed to estimate the false discovery rate (FDR) of phosphosites based on available evidence in PSP and/or PA and predict a more realistic count of true phosphosites. We ranked sites into phosphorylation likelihood sets based on layers of accumulated evidence and then analysed them in terms of amino acid conservation across 100 species, sequence properties and functional annotations of associated proteins. We demonstrated significant differences between the sets and developed a method for independent phosphosite FDR estimation. Remarkably, we estimated a false discovery rate of 86.1%, 95.4% and 82.2% within sets of described phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) sites respectively for which only a single piece of identification evidence is available (the vast majority of sites in PSP). Overall, we estimate that [~]56,000 Ser, 10,000 Thr and 12,000 Tyr phosphosites in the human proteome have truly been identified to date, based on evidence in PSP and/or PA, which is lower than most published estimates. Furthermore, our analysis estimated [~]91,000 Ser, 49,000 Thr and 26,000 Tyr sites that are likely to represent false-positive phosphosite identifications. We conclude that researchers should be aware of the significant potential for false positive sites to be present in public databases and should evaluate the evidence behind the phosphosites used in their research.

Source connections

Explore related subjects

bioinformaticsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kalyuzhnyy, A., Eyers, P. A., Eyers, C. E., Sun, Z., Deutsch, E. W., Jones, A. R.. 2021-04-15. Profiling the Human Phosphoproteome to Estimate the True Extent of Protein Phosphorylation. https://doi.org/10.1101/2021.04.14.439901

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

KEEP EXPLORING

Related preprints

bioRxiv2026

spatialMET: an open and scalable framework for spatial metabolomics analysis

Mass spectrometry imaging (MSI) enables spatially resolved metabolomics in intact tissue sections, but analysis remains challenging at scale. Existing MSI workflows often require users to combine multiple software tools, while others rely on proprietary vendor software that limits interoperability and reproducibility. To address these challenges, we developed spatialMET, an open-source framework that provides an end-to-end workflow for MSI analysis. spatialMET provides a unified platform for preprocessing, spatial domain detection, and visualization. Downstream analyses include differential abundance testing, spatial autocorrelation and gradient analysis, dimensionality reduction, and correlation network analysis. Spatial domain detection uses hcdist, a C-based hierarchical clustering implementation that substantially reduces runtime and memory use relative to existing R-based approaches. spatialMET can be run through an interactive R Shiny application or as a standalone command-line workflow for larger datasets or high-performance computing environments. Applied to mouse small cell lung cancer MALDI-MSI data containing 284,673 pixels, spatialMET identified tumor-associated, stromal, and adjacent lung spatial domains that aligned with matched histology. Differential abundance analysis identified 117 m/z features that differed between tumor and stromal regions, while spatial autocorrelation analyses revealed spatially structured abundance patterns. Applying spatialMET to mouse lung adenocarcinoma data from an entire lung lobe containing 338,477 pixels further demonstrated scalability and captured spatial heterogeneity across tumor and surrounding lung tissue. In summary, spatialMET provides a scalable, open-source framework for end-to-end spatial metabolomics analysis, and it is distributed as a Docker container for reproducible deployment. Source code and installation instructions are available at https://github.com/biodatalab/spatialMET.

bioinformatics↗
bioRxiv2026

Probing the transcriptome response to shivering in skeletal muscle using a multilayered bioinformatics approach

Cold acclimation holds therapeutic potential for improving metabolic health. We previously demonstrated that repeated cold-induced shivering enhances insulin sensitivity in humans. However, the molecular pathways that underlie the skeletal muscle shivering response, and how these relate to beneficial physiological effects, remain poorly understood. In this study, we combined complementary bioinformatics approaches to allow in-depth analysis of the transcriptomic response of human skeletal muscle to repeated shivering. We identified a robust transcriptional signature and show a sex-specific component in the shivering skeletal muscle response, which seemed to diminish following cold adaptation. Our findings provide mechanistic insights into cold-induced muscle adaptations, shed light on potential interesting molecular targets for further investigation, and emphasize the importance of including both sexes in future cold acclimation studies.

bioinformatics↗
bioRxiv2026

An Information Geometry approach to model topological trajectories and Gene Expression Radius from UMAP geometry.

Understanding the relationship between gene expression dynamics and cellular identity remains a central challenge in single cell biology. Here, we introduce a novel computational and mathematical framework that integrates information geometry, fuzzy topology, and UMAP analysis to model gene expression landscapes derived from single cell RNA sequencing data. We formalize gene expression data as a fuzzy topological space, where interactions between expression points are governed by probabilistic distributions inspired by manifold learning approaches such as UMAP. Within this framework, we define an information geometric structure through a Fisher metric induced by these distributions, enabling the computation of geodesic trajectories that capture cellular differentiation processes. A key contribution of this work is the derivation of analytical conditions, expressed as expression radius formulas, that characterize local neighborhoods in gene expression space. These conditions allow for the identification of genes associated with stem cell states and predictions in transitional cell types in future work. Application of the proposed framework to single cell datasets reveals biologically meaningful gene sets enriched in key regulatory pathways and transcription factors, demonstrating the capacity of our approach to uncover latent structure in complex gene expression data. Our results suggest that integrating differential geometry with statistical learning theory offers a powerful paradigm for modeling genotype and phenotype relationships and cellular state transitions, with potential implications for precision medicine and systems biology.

bioinformatics↗