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.
