title: Highly sensitive phosphoproteomics by tailoring solid-phase extraction to electrostatic repulsion-hydrophilic interaction chromatography.
abstract: In the past decade, several strategies for comprehensive phosphoproteome analysis have been introduced. Most of them combine different phosphopeptide enrichment techniques and require starting material in the milligram range, as a consequence of their insufficient sensitivity. This limitation impairs the applicability of phosphoproteomics to a wide variety of clinical research, where sample material is highly limited. Here we introduce a highly sensitive and easy-to-establish 2D bottom-up strategy for microgram-scale phosphoproteomics, based on electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), a simple solid-phase extraction step by strong cation exchange (SCX) or reversed phase (RP), and LC-MS analysis. With only 100 μg of tryptic digested, nonstimulated HeLa protein and 45 h of LC-MS analysis time, we identified ≥7500 nonredundant and highly confident phosphorylation sites (per replicate). We assigned all phosphorylation sites to 3013 phosphoproteins, covering the entire dynamic range from 10(7) down to a few copies per cell. Compared to affinity-based-enrichment methods using Ti(4+), our ERLIC-based strategy enriched considerably longer and more acidic phosphopeptides and consequently, we identified 327 phosphorylated C-terminal peptides. The simplicity and high sensitivity of ERLIC-SCX/RP-LC-MS render its future promising for microgram-scale-phosphoproteomics in biological, biomedical, and clinical research.
authors.affiliation: Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. , Otto-Hahn-Straße 6b, 44227 Dortmund, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Loroch
authors.forename: René Peiman
authors.initials: RP
authors.lastname: Zahedi
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
mesh_terms: D002852:Chromatography, Ion Exchange / Q000379:methods*; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D010748:Phosphopeptides / Q000032:analysis* / Q000302:isolation & purification; D010750:Phosphoproteins / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D052616:Solid Phase Extraction / Q000379:methods*; D055672:Static Electricity; D053719:Tandem Mass Spectrometry
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins