Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides

Tóth, Gábor and Sugár, Simon and Balbisi, Mirjam and Molnár, Balázs András and Bugyi, Fanni and Fügedi, Kata Dorina and Drahos, László and Turiák, Lilla (2022) Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides. MOLECULES, 27 (19). No-6645. ISSN 1420-3049

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Abstract

The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures.

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