Comparison of solid-phase extraction methods for efficient purification of phosphopeptides with low sample amounts

Efficient phosphoproteomic analysis of small amounts of biological samples (e.g. tissue biopsies) requires carefully selected enrichment and purification steps prior to the nanoflow HPLC-MS/MS analysis. Solid -phase extraction (SPE) is one of the most commonly used approaches for sample preparation. Several stationary phases are available for peptide SPE purification, however, most of the published methods are not optimized to provide good recoveries of phosphorylated peptides. Our goal was to investigate the performance of 13 self-packed and 3 commercial centrifugal SPE cartridges/spin tips, thus enhanc-ing the efficiency of the phosphoproteomic analysis of small amounts of complex protein mixtures. Eight reversed-phase (RP), five graphite, two ion-exchange, and one hydrophilic-lipophilic balance (HLB) sta-tionary phase were evaluated. Two RP, one graphite, and the HLB self-packed centrifugal SPE tips pro-vided excellent results for the purification of 1 mu g tissue and cell line digests. Using these methods, the sample loss was significantly reduced compared to one of the commercial SPE methods, 22-58% more unique phosphopeptides were identified, and the recovery was higher by 132-155%. (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Automated summary

Efficient phosphoproteomic analysis of small amounts of biological samples requires careful enrichment and purification steps. This study evaluated the performance of various self-packed and commercial centrifugal SPE cartridges/spin tips, and identified several methods that provided excellent results in terms of sample purification, reducing sample loss, and increasing the detection and recovery of phosphopeptides.