Comprehensive and Reproducible Phosphopeptide Enrichment Using Iron Immobilized Metal Ion Affinity Chromatography (Fe-IMAC) Columns

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 g 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. Molecular & Cellular

Comparing Multistep Immobilized Metal Affinity Chromatography and Multistep TiO2 Methods for Phosphopeptide Enrichment

“…Phosphopeptides need to be enriched prior to mass spectrometric analysis to reduce the sample complexity and minimize the repression effects from non-phosphorylated peptides 4-6 . There are several approaches for enriching phosphopeptides from the peptide mixtures, including immobilized metal affinity chromatography (IMAC) with Fe 3+ or Ti 4+ 7,8,9 , metal oxide affinity chromatography (MOAC) with TiO 2 10,11 , and immuno-affinity based methods 12 . Other methods such as polymer-based metal ion affinity capture (PolyMAC) 13,14 and polymer-oxotitanium-modified gold wafer (Au-P-oxoTi) 15 result in successful phosphopeptide enrichment.…”

ProteomeTools: Systematic Characterization of 21 Post-translational Protein Modifications by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) Using Synthetic Peptides

“…This makes the technique more generic than antibody-based detection as it does not rely on the generation of specific reagents for each and every case. In general, PTMs are studied at the level of peptides following protease digestion of full proteomes and the modified peptides may be subjected to specific chromatographic, chemical or antibody based enrichment steps prior to LC-MS/MS analysis to overcome issues associated with the often low stoichiometry of the PTM (3)(4)(5). One of the key steps in the analysis workflow is searching the tandem mass spectra against an in-silico digested database of protein sequences.…”

Recent advances in phosphoproteomics and application to neurological diseases

“…To mitigate the variation and sample loss from each manual enrichment step, the automated HPLC column 57 and cartridge platform for Fe 3+ -IMAC 58 have been developed. The benefit of column-based enrichment is that the performance is not perturbed by the bead-to-sample ratio and variation from manual steps.…”

“…79 Due to the high selectivity of phosphopeptide enrichment, fractionation of phosphopeptides followed by phosphopeptide enrichment, often called the “enrichment-fractionation” approach, is prevalent in a variety of recent reports. 57, 79, 80 …”