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Comparison of solid-phase extraction methods for efficient purification of phosphopeptides with low sample amounts.
Fanni Bugyi1, Gábor Tóth2, Kinga Bernadett Kovács3
1MS Proteomics Research Group, Research Centre for Natural Sciences, Magyar tudósok körútja 2, 1117 Budapest, Hungary; Hevesy György PhD School of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/a, 1117 Budapest, Hungary.
Journal of Chromatography. A
|November 13, 2022
Summary
Optimizing solid-phase extraction (SPE) for phosphoproteomic analysis is crucial for small samples. Self-packed SPE tips significantly improve phosphopeptide recovery and identification in complex mixtures.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Phosphoproteomic analysis of limited biological samples necessitates efficient enrichment and purification.
- Solid-phase extraction (SPE) is a common sample preparation technique, but existing methods often lack optimization for phosphopeptide recovery.
- Limited sample amounts, such as tissue biopsies, pose challenges for comprehensive phosphoproteomic studies.
Purpose of the Study:
- To evaluate the performance of various self-packed and commercial centrifugal SPE cartridges for phosphopeptide purification.
- To enhance the efficiency of phosphoproteomic analysis from small, complex biological samples.
- To identify optimal SPE stationary phases for maximizing phosphopeptide recovery and identification.
Main Methods:
- Evaluation of 13 self-packed and 3 commercial centrifugal SPE cartridges/spin tips.
- Testing of eight reversed-phase (RP), five graphite, two ion-exchange, and one hydrophilic-lipophilic balance (HLB) stationary phases.
- Analysis of 1 µg tissue and cell line digests using nanoflow HPLC-MS/MS.
Main Results:
- Two RP, one graphite, and the HLB self-packed centrifugal SPE tips demonstrated excellent purification performance.
- These optimized methods significantly reduced sample loss compared to a commercial SPE method.
- An increase of 22-58% in unique phosphopeptides identified and 132-155% higher recovery were achieved.
Conclusions:
- Self-packed centrifugal SPE tips, particularly specific RP, graphite, and HLB phases, are highly effective for phosphopeptide enrichment from small samples.
- Optimized SPE protocols substantially improve phosphoproteomic analysis efficiency, yielding more identified phosphopeptides and higher recovery rates.
- These findings provide a foundation for more sensitive and comprehensive phosphoproteomic studies using limited biological material.
