PROJECT SUMMARY Molecular analysis of limited biological and clinical samples is of high significance and growing interest, as observed from the exponentially increasing number of publications, citations, and startup businesses. Scarce, precious, and amount-limited clinical and biological samples, including small-volume liquid biopsies (i.e., blood, CSF, and other physiological fluids), microneedle tissue biopsies, rare cell isolates (e.g., circulating tumor cells, cells from liquid biopsies, or laser-capture microdissected tissue biopsies), neonatal specimens, model animal samples, dried blood spots, minute amounts of cells used in cell-based therapies, and even single cells and organelles often hold keys for solving long-standing puzzles in biomedical research. Currently, the most efficient technologies to tackle such limited samples rely on transcriptomic and genomic profiling techniques. Unfortunately, genomic and transcriptomic profiles are poor surrogates for predicting quantitative proteomic profiles, and these techniques cannot deliver structural and quantitative information for the characterization of proteoforms, post-translational modifications (PTMs), and protein interactions, which are essential for discovering cellular and molecular level pathways of disease, mechanisms of cell signaling, cell activation/differentiation states, as well as novel biomarkers and therapeutic targets. Therefore, the ability to reliably characterize proteomes in such limited samples will help define strategies for enabling early diagnostics and personalized treatments of deadly diseases (e.g., cancer, Alzheimer’s disease, infectious diseases, cardiovascular diseases, and traumatic brain injury), ensure the efficiency of cell-based therapies, characterize cell model systems, and answer pressing questions in fundamental biology that could not be resolved before. However, deep and high-sensitivity proteomic profiling of limited samples using conventional mass spectrometry (MS)-based proteomic techniques is still a major challenge because (1) there are no amplification techniques available for proteins, proteoforms, and PTMs, and (2) the current state-of-the-art conventional nanoflow liquid chromatography (nanoLC) technologies provide suboptimal sensitivity and separation performance for scarce samples. Consequently, numerous critical biological and pathological phenomena can not be investigated by research laboratories using amount-limited samples, and multiple deadly diseases can not be diagnosed early and successfully treated. In this study, we propose a unique ultra-low flow (ULF) proteomic technology based on porous layer open-tubular (PLOT) and monolithic nanoLC columns developed by our laboratory that has the potential to disrupt a sector of the nanoLC products market to enable deep proteomic profiling of scarce biological and clinical samples that would be critical for numerous academic, clinical diagnostics, and forensic research laboratories, as we...