A Reproducible Multichannel Synchronous Rapid Phosphopeptide Enrichment Strategy Enabled by Anisotropic Porous Chitosan-Based Arrays

Large-scale protein phosphorylation analysis has become a mainstream approach for investigating tumor biomarkers and drug targets. However, the cumbersome processing procedures associated with hundreds of clinical samples and intrinsic batch-to-batch variations render this work highly challenging. Herein, we designed an anisotropic porous monolith, CS-ZrPC@PLP, based on directional freeze-casting and constructed an enrichment array to establish a high-throughput analytical method for complex biosamples. The fabricated monolith forms highly parallel capillary channels along the ice crystal growth direction, enabling low mass transfer resistance and excellent mechanical stability to abundantly expose active sites and ensure pressure tolerance during high-throughput enrichment operations. Benefiting from these features, a multichannel synchronous rapid enrichment (MCSR) strategy was proposed without the frequent centrifugation steps required in traditional enrichment methods. Simultaneous phosphopeptide enrichment of eight sample sets, including tryptic digests of standard protein mixtures and complex biological samples such as cell lysates, was accomplished within 5 min using only aspiration-dispersion cycles in the MCSR strategy, whereas traditional methods typically require tens of minutes to hours for one sample. Enrichment results demonstrated that the MCSR strategy exhibited significant selectivity for phosphopeptides even in the presence of 1000-fold BSA interference (molar ratio of BSA/β-Casein, 1000/1). Notably, despite using microflow LC-MS/MS analysis rather than nano-LC-MS/MS, 41,626 phosphopeptides and 7,168 phosphoproteins were successfully identified from Hep G2 cell digests after 5 min of enrichment. Furthermore, the MCSR strategy demonstrates excellent compatibility with commercial robotic pipetting platforms, highlighting the great potential for rapid, high-throughput phosphopeptide enrichment in clinical proteomics research.