Authors: Shuang Zhang | Xianting Ding
Source: Chinese Science Bulletin (Chinese Version) (article, 2025-02-28)
Field: Chemistry — Inorganic Chemistry

Trace sample proteomics pre-treatment technology based on boric acid metal-organic frameworks <sec><p indent="0mm">Proteins are the most information-rich biomolecules which are essential for normal biochemical functions and implicated in various disease processes. Mass spectrometry (MS)-based label-free proteomics has emerged as a crucial technique for protein identification. In bottom-up proteomic analysis, the effective acquisition of data necessitates the digestion of proteins extracted from complex biological samples into pure peptides. The Filter Aided Sample Preparation (FASP) workflow is widely regarded as the gold standard for this process. However, conventional FASP workflow involves multiple redundant steps, such as repeated pipetting, rinsing, and ultrafiltration, which can result in substantial protein loss due to non-specific adsorption and contamination. This issue is particularly critical when dealing with limited and often non-renewable clinical samples, including rare cell subsets and small volumes of blood or cerebrospinal fluid. </sec><sec> To address these challenges, various nanoparticles have been developed to capture proteins through nano-bio interactions in clinical samples before mass spectrometry (MS) acquisition for comprehensive proteomic analysis. The utilization of surface-functionalized magnetic nanoparticles, in conjunction with robotic nanoliter liquid handling systems, enables rapid and high-throughput proteomic analysis. Despite these advancements, the manufacturing of compatible automated instruments remains a significant barrier to accessibility. The design of functional materials for the enrichment of trace proteins in single cells and tissue sections is of great importance and remains a persistent challenge. </sec><sec> Protein surfaces are usually occupied by cationic groups such as amine, imidazole, guanidinium, and anionic carboxylate groups. Hence, developing functional materials that are capable of embracing both the anionic and cationic groups on the protein surface could effectively entrap proteins. Phenylboronic acid (PBA) is a highly electron-deficient group that could bind with electron-rich amino acid residues such as amine on lysine and imidazole on histidine via nitrogen-boronate coordination. The aromatic ring in PBA allows interaction with the guanidinium or ammonium groups on proteins via cation-π interactions. Metal-organic frameworks (MOFs) are formed through the self-assembly of ligands and metal ions, enabling coordination with the free carboxyl groups of proteins. Developing novel MOFs that are designed to capture entire proteins to profile the whole-proteomics in trace samples is highly significant but has been overlooked. </sec><sec> Herein, we synthesized a class of boronic acid-rich lanthanide metal-organic frameworks (MOFs) using 3,5-dicarboxyphenylboronic acid and lanthanide metal salts. The thus manufactured MOFs with high boronic acid grafting ratios enable high binding affinity with different types of proteins via a combination of nitrogen-boronate complexation, cation-π interaction, and coordination between the abundant open metal sites and the bare amino acids residues on the protein surface. We further developed a MOFs Aided Sample Preparation (MASP) pipeline for deep proteome of trace biological samples. Specifically, the MOFs were loaded and proteins in biological samples were captured, followed by discarding the supernatant containing impurities through centrifugation. Subsequently, the proteins on the surface of MOFs were rinsed and enzymatically digested into peptides, which were eluted and desalted before MS acquisition. The MASP process streamlines the protein sample preparation steps by integrating lysis, washing, and digestion within a single PCR tube, thereby reducing sample loss due to surface adsorption and enhancing the depth of proteomic identification in ultra-trace samples. We evaluated the MASP method with 10 to 5000 HEK 293T cells and demonstrated the feasibility and efficiency of MASP in processing trace cells. Our results indicate that MASP enables label-free proteomic analysis in as few as 10 HEK-293T cells, making in-depth proteome analysis more feasible and robust in practical clinical scenarios. </sec> Computer science Metal-Organic Frameworks: Synthesis and Applications ; Advanced Proteomics Techniques and Applications ; Advanced Nanomaterials in Catalysis