Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns*

By M. Larsen, T. Thingholm, Ole Nørregaard Jensen, P. Roepstorff, T. Jørgensen. Molecular & Cellular Proteomics, 2005

1,438 citations recorded by the source corpus.

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/979b1ee6d8c32378c1182b3d393de33d7260e9f2

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Citations in the corpus

Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle. (2008, methodology): “Resulting peptide fractions were subjected to phosphopeptide enrichment on TiO2 beads prior to analysis by nanoscale liquid chromatography-tandem MS (LC-MS/MS) on a high-resolution linear ion trap/orbitrap (LTQ-Orbitrap) hybrid mass spectrometer (Larsen et al., 2005; Olsen et al., 2006) (Figure 2A).”
Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis*S (2007, methodology): “Subsequently each fraction was separately enriched for phosphopeptides using TiO2 affinity chromatography (23) in the presence of high concentrations of dihydroxybenzoic acid ( 36 ).”
Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips (2007, methodology): “Phospho-affinity beads using immobilized metal affinity materials or metal oxides chromatography (MOC), titania (titanium dioxide) and zirconia (zirconium dioxide) are packed on reversed-phase disks in pipette tips, allowing fast enrichment protocols of well below 1”
Mapping protein post-translational modifications with mass spectrometry (2007, background): “Titanium dioxide (TiO 2 ) and zirconium dioxide (ZrO 2 ) are also used to adsorb phosphopeptides, through bidentate interaction”
Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. (2006, methodology): “Protein digestion was performed essentially as in Foster et al. (2003), strong-cation exchange chromatography of digests as in Gruhler et al. (2005), and TiO2 enrichment of phosphopeptides essentially as in Larsen et al. (2005).”
Proteomics by mass spectrometry: approaches, advances, and applications. (2009, background): “By limiting nonspecific interactions, 2,5-dihydroxybenzoic acid (DHB) (204) and aliphatic hydroxyl acids (205) increase selectivity of TiO2.”