Nano-liquid chromatography with monolithic stationary phase based on naphthyl monomer for proteomics analysis

• Published in: Journal of Chromatography A Vol. 1690; p. 463804
• Main Authors: Aydoğan, Cemil, Beltekin, Büşra, Alharthi, Sarah, Ağca, Can Ali, Erdoğan, İbrahim Y.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.02.2023
• Subjects: Chromatography, Liquid - methods; Cytochromes c; Miniaturization; Monolith; Nano-LC; Proteomics; Vinyl Compounds - chemistry; Vinylnaphthalene; Miniaturization; Nano-LC; Monolith; Vinylnaphthalene; Proteomics
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2023.463804

•2-vinylnaphthalene (2-VNA) based monolithic column with 50 µm i.d. was specially designed for nano-LC.•The 2-VNA column showed good selectivity toward aromatic compounds.•Intact proteins were readily separated on the 2-VNA monolithic column.•Peptide separation of cyt c with high peak capacity of 1440 was demonstrated.•Proteomics analysis of COS-7 (CV-1 in origin with SV40 genes) cell line was demonstrated. Monolithic poly(2-vinylnaphthalene-co-divinylbenzene) columns were introduced, for the first time, and were evaluated as the separation media for nano-liquid chromatography (nano-LC). These columns were prepared by in-situ polymerization of 2-vinylnaphthalene (2-VNA) as the functional monomer and divinylbenzene (DVB) as the crosslinker in a fused silica capillary column of 50 µm i.d. Various porogenic solvents, including tetrahydrofuran (THF), dodecanol and toluene were used for morphology optimization. Final monolithic column (referred to as VNA column) was characterized by using scanning electron microscopy (SEM) and chromatographic analyses. Alkylbenzenes (ABs), and polyaromatic hydrocarbons (PAHs) were separated using the VNA column while the column offered excellent hydrophobic and π-π interactions under reversed-phase conditions. Theoretical plates number up to 41,200 plates/m in isocratic mode for ethylbenzene could be achieved. The potential of the final VNA column was demonstrated with a gradient elution in the  separation of six intact proteins, including ribonuclease A (RNase A), cytochrome C (Cyt C), lysozyme (Lys), β-lactoglobulin (β-lac), myoglobin (My) and α-chymotrypsinogen (α-chym) in nano LC system. The column was then applied to the peptide analysis of trypsin digested cytochrome C, allowing a high peak capacity up to 1440 and the further proteomics analysis of COS-7 cell line was attempted applying the final monolithic column in nano-LC UV system.