Cross-linking mass spectrometry for structure analysis of the intrinsically disordered Tau and phosphorylated Tau protein
Cristian Arsene, Alexander Gates, Anne-Katrin Römmert, André Märtens, Valentina Faustinelli, Luise Luckau, Gavin O’Connor
Abstract
We present a novel method for analyzing the folding of intrinsically disordered proteins (IDPs), such as Tau and phosphorylated Tau (pTau), in solution. Using cross-linking mass spectrometry (XL-MS) combined with a new downstream analysis framework, we construct weighted interaction networks from cross-link–derived residue pairs without relying on predefined secondary structure assumptions.
Introduction
Structural characterization is particularly challenging for intrinsically disordered proteins, whose conformations often cannot be resolved by conventional techniques such as X-ray crystallography or cryo-EM. In these cases, alternative approaches are required to probe three-dimensional organization, even if only at moderate spatial resolution.
Methods
Cross-link detection saturation analysis across replicated measurements. To assess cross-linking site coverage across measurements, replicates of datasets were randomly permuted and processed to extract non-homeotypic cross-links. After filtering, site-pairs were converted to unique combinations and counted cumulatively over increasing numbers of technical replicates.
Results and discussion
Identification of cross-links and distance-constraints.
The analytical workflow commenced with a cross-linking reaction followed by trypsin digestion of the modified protein. Building upon an established XL-MS protocol [3], liquid chromatographic conditions were further refined to enhance the reliable detection of cross-linked peptides across a larger number of technical replicates.
Conclusion
The new cross-linking analysis method enables the sensitive detection and relative quantification of structural divergence between protein conformers under different conditions and over time. This opens the possibility to further investigate the influence of post-translational modification (PTM) patterns e.g. the differential phosphorylation of Tau on the relationship between structure and aggregation.
Citation: Arsene C, Gates A, Römmert A-K, Märtens A, Faustinelli V, Luckau L, et al. (2026) Cross-linking mass spectrometry for structure analysis of the intrinsically disordered Tau and phosphorylated Tau protein. PLoS Comput Biol 22(1): e1013868. https://doi.org/10.1371/journal.pcbi.1013868
Editor: Arli Aditya Parikesit, Indonesia International Institute for Life Sciences, INDONESIA
Received: September 18, 2025; Accepted: December 22, 2025; Published: January 14, 2026
Copyright: © 2026 Arsene et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The mass spectrometry proteomics data and scripts have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD067659.
Funding: This research has received funding from the European Partnership on Metrology, cofinanced from the European Union’s Horizon Europe Research and Innovation Programme, and by the Participating States (European Partnership on Metrology, 10.13039/100019599, 22HLT07 NEuroBioStand to CA, AKR, VF and LL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
