Nano-LC with New Hydrophobic Monolith Based on 9-Antracenylmethyl Methacrylate for Biomolecule Separation

In this study, new monolithic poly(9-anthracenylmethyl methacrylate-co-trimethylolpropane trimethacrylate (TRIM) columns, referred as ANM monoliths were prepared, for the first time, and were used for the separation media for biomolecules and proteomics analysis by nano-liquid chromatography (nano-LC). Monolithic columns were prepared by in situ polymerization of 9-anthracenylmethyl methacrylate (ANM) and trimethylolpropane trimethacrylate (TRIM) in a fused silica capillary column of 100 µm ID. Polymerization solution was optimized in relation to monomer and porogenic solvent. Scanning electron microscopy (SEM) and chromatographic analyses were performed for the characterization studies of ANM monoliths. The ANM monolith produced more than 46.220 plates/m, and the chromatographic evaluation of the optimized ANM monolith was carried out using homologous alkylbenzenes (ABs) and polyaromatic hydrocarbons (PAHs), allowing both strong hydrophobic and π-π interactions. Run-to-run and column-to-column reproducibility values were found as <2.91% and 2.9-3.2%, respectively. The final monolith was used for biomolecule separation, including both three dipeptides, including Alanine-Tyrosine (Ala-Tyr), Glycine-Phenylalanine (Gly-Phe), and L-carnosine and five standard proteins, including ribonuclease A (RNase A), α-chymotrypsinogen (α-chym), lysozyme (Lys), cytochrome C (Cyt C), and myoglobin (Mb) in order to evaluate its potential. Both peptides and proteins were baseline separated using the developed ANM monolith in nano-LC. The ANM monolith was then applied to the protein and peptide profiling of MCF-7 cell line, which allowed a high-resolution analysis of peptides, providing a high peak capacity.