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

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 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.