Arsene, C. | bioRxiv Science

bioRxiv2025

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.

biochemistry↗

bioRxiv2023

Label-free Quantification of Host-Cell Protein Impurity in a Recombinant Hemoglobin Reference Material

Quantitative analysis depends on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host-cell proteins (HCPs) in bioengineered proteins. For example, hemoglobin-A2 (HbA2) is being used as obtained through overexpression in E.coli. Two different materials had been produced: natural, and U-15N-labeled HbA2. For quantification of impurity, precursorion (MSl-) intensities were integrated over all E.coli -proteins identified, and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for E.coli-cell lysate, which had been spiked at known mass-ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results could also be obtained when the complexity of the calibrator is reduced to a mix of nine proteins, and a minimum of five proteins is estimated to be sufficient to keep the sampling error below l5%. For the studied materials, HbA2-mass fractions of 916{+/-}15 mg/g and 922{+/-}11 mg/g were found. Value assignment by LFQ thus contributes 1-2% to the overall uncertainty of HbA2-quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded 999.1{+/-} 0.15 mg/g, corresponding to {approx} 0.2% of uncertainty contribution, though at a significant loss of material. If an overall-uncertainty of 5% is acceptable for protein-quantification, working with the original materials would definitely be viable, therefore.

biochemistry↗