Abstract
Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO(2), Ti, and Fe immobilized metal ion affinity chromatography (IMAC) material in batch or microtip format are widely used, but they suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichment in an HPLC column format. We found that Fe-IMAC columns enabled the selective, comprehensive, and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100 μg to 5 mg of digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median cv of 15%). The number of unique phosphopeptides could be increased to more than 14,000 when the IMAC eluate was subjected to a subsequent hydrophilic strong anion exchange separation. Fe-IMAC columns outperformed Ti-IMAC and TiO(2) in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow-throughs showed that all materials largely bound the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution, and the stochastic nature of data-dependent acquisition in mass spectrometry are the causes of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis that can be applied to a wide range of biological systems.
Highlights
From the * Chair of Proteomics and Bioanalytics, Technische Universitat Munchen, Emil Erlenmeyer Forum 5, 85354 Freising, Germany, ¶ Center for Integrated Protein Science Munich, Emil Erlenmeyer Forum 5, 85354 Freising, Germany
Chromatographic Phosphopeptide Enrichment Using an Fe-IMAC Column—As discussed above, batch and tip-based enrichment protocols have been found to suffer from irreproducibility due to a number of factors including significant manual intervention
We conducted a series of experiments that supported this hypothesis, and the data collectively show that these practical issues can be substantially reduced by the use of a conventional FeIMAC HPLC column (Fig. 1)
Summary
From the * Chair of Proteomics and Bioanalytics, Technische Universitat Munchen, Emil Erlenmeyer Forum 5, 85354 Freising, Germany, ¶ Center for Integrated Protein Science Munich, Emil Erlenmeyer Forum 5, 85354 Freising, Germany. At least one-third to one-half of all human proteins are thought to be phosphorylated at some point [2, 3], and hundreds of thousands of different phosphorylation sites are believed to exist Because of this molecular complexity and the often substoichiometric extent of phosphorylation, specific enrichment prior to analysis via liquid chromatography– tandem mass spectrometry (LC-MS/MS) is generally required [4, 5]. Most current such strategies exploit the affinity of phosphate groups for metals immobilized on carrier resins. It turns out that current mass spectrometers still lack the scan speed and dynamic range required to reach complete (phospho)peptide sampling in direct LC-MS/MS measurements [36, 37]
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