A simplified one-step chaotropic extraction workflow for quantitative proteomic analysis of highly mineralized...

Proteomic analysis of highly mineralized biological matrices remains analytically challenging due to the dense inorganic framework that restricts solvent accessibility and strongly binds proteins within the mineral phase. In this study, we systematically evaluated 16 protein extraction strategies combining different demineralization approaches (EDTA, HCl, or no demineralization) with four extraction buffer systems (guanidine hydrochloride, urea/thiourea, SDS, and Extraction Reagents Type 4 (ERT4)). Using cow femur bone as a model mineralized matrix, protein recovery was assessed by quantifying both demineralization supernatants and residual pellets, complemented by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Among all tested conditions, conventional two-step protocols often led to significant protein redistribution and loss during demineralization. A simplified one-step strategy combining ERT4 with guanidine hydrochloride (ERT4/GuHCl) was developed and achieved a comparable protein yield and identified the highest number of unique protein groups with broad coverage across isoelectric point, molecular mass, and hydropathy distributions. Analytical evaluation confirmed, that this optimized strategy maintained high digestion quality and preserved protein integrity. The optimized workflow was transferred to coralline red algae (CRA), an unconventional, extremely mineralized biological sample. The protocol allowed for broad physicochemical coverage despite biomass and database limitations. Overall, this work highlights the limitations of conventional demineralization-based workflows, and positions ERT4/GuHCl as an efficient and chemically safe strategy for proteomic analysis of highly mineralized matrices, such as bone and coralline red algae.