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

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

Blog Article

The optimization of solid-phase extraction (SPE) Throw 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 Kit DIY analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures.