# Cross-linking mass spectrometry for structure analysis of the intrinsically disordered Tau and phosphorylated Tau protein

## TL;DR

This paper introduces a new method using cross-linking mass spectrometry to study the structure of disordered proteins like Tau and phosphorylated Tau, revealing how they change shape in solution.

## Contribution

A novel XL-MS-based framework is introduced for analyzing IDP folding without assuming secondary structures, enabling structural comparisons of Tau and pTau.

## Key findings

- Weighted interaction networks from cross-link data reveal structural differences between protein conformations.
- At least 500 cross-links are needed for reliable detection of structural divergence in BSA.
- Tau and pTau show distinct structural evolution patterns during aggregation induced by arachidonic acid.

## 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. Structural differences between protein conformations are quantified by comparing the organization of loop structures within their cross-link networks. Validation with bovine serum albumin (BSA) in native and denatured states shows that at least 500 cross-links—requiring 5–10 replicate measurements—are needed for reliable detection of structural divergence. Leave-one-out analysis confirms that structural transitions are global, highlighting the importance of comprehensive cross-link datasets. The coverage of unique cross-links was evaluated using accumulation curves from randomized permutations. Saturation levels were found to be 9.7%, 5.0%, and 6.2% of the total 528 and 10,731 possible cross-links after 30, 84, and 62 technical replicates, respectively, for myoglobin, native BSA, and denatured BSA. For Tau and pTau, coverage reached 10.8% and 5.5% of the upper limit (8,256). Finally, applying our structural analysis to Tau and pTau during arachidonic acid–induced aggregation revealed distinct patterns of structural evolution between the two proteins.

Intrinsically disordered proteins (IDPs) play key roles in cellular regulation and disease, yet their structural characterization remains challenging due to their dynamic and non-canonical conformations. In this study, we present a novel approach for analyzing the folding behavior of IDPs in solution, using Tau and phosphorylated Tau (pTau) as model systems. By refining an existing cross-linking mass spectrometry (XL-MS) protocol and developing a downstream analysis pipeline, we constructed weighted interaction networks from cross-link–derived residue pairs without relying on predefined secondary structure assumptions. These networks are transformed into loop-based structural maps that represent conformational states, enabling their unsupervised clustering and pairwise comparison. Validation with native and denatured bovine serum albumin (BSA) demonstrates the method’s sensitivity to global structural transitions. We further evaluated cross-link coverage saturation using randomized accumulation curves for myoglobin, BSA, and denatured BSA. Applying this framework to Tau and pTau during arachidonic acid–induced aggregation reveals distinct patterns of structural evolution. The method allows for moderate-resolution structural insights into IDPs, which are otherwise inaccessible by traditional techniques such as X-ray crystallography or cryo-EM.