Unlocking Ultrasensitive Phosphoproteomics of Nanoscale Samples: A Rapid IMAC Enrichment Platform Using Ti-PAN Tip

Protein phosphorylation is a critical mechanism in cellular signal transduction, playing essential roles in various cellular processes. However, significant challenges of their low abundance, high dynamic range, and complexity are especially prominent for accurate phosphoproteome analysis in nanoscale samples, such as oocytes, where misregulation of phosphorylation can lead to infertility. In this study, we developed a novel phosphopeptide enrichment tip (Ti-PAN) using highly hydrophilic textile PAN fibers, enabling rapid in-situ one-tip IMAC enrichment in just 8 minutes. This approach not only minimizes sample loss but also enhances analytical sensitivity and is easily adaptable to automation for high-throughput applications, demonstrating excellent recovery (>94%) and outstanding selectivity for phosphopeptides with remarkable resistance to interference up to 1:5000. By integrating it with a compatible sample preparation workflow based on n-dodecyl {beta}-D-maltoside (DDM) lysis with the high-sensitivity MS data acquisition method of turboDDA, comprehensive mapping of phosphosites in microsamples was achieved, including those from the ERK interactome, mouse brain slices, and oocytes. Using this approach, we identified 2,139 to 11,296 phosphosites across 645 to 2,168 phosphoproteins from samples ranging from 100 to 100,000 cells. Furthermore, we successfully applied this strategy to analyze the phosphoproteome of mouse oocytes at different developmental stages, using as few as 5 oocytes per sample. This led to the identification of 2,709 phosphosites on 701 phosphoproteins, revealing several key kinases involved in meiotic resumption and oocyte maturation, including STK4 (serine/threonine kinase 4) and DYRK3 (dual specificity tyrosine phosphorylation-regulated kinase 3). To our knowledge, this represents the deepest coverage of phosphoproteomes from such minimal oocyte material reported to date, offering valuable mechanistic insights into developmental controls during oocyte maturation. This Ti-PAN-based method provides a robust, versatile, and automatable platform for large-scale, highly sensitive phosphopeptide enrichment, substantially advancing phosphoproteomic studies in complex biological systems.