Comparison of ERLIC–TiO₂, HILIC–TiO₂, and SCX–TiO₂ for Global Phosphoproteomics Approaches

Abstract

Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.

Characterization of early autophagy signaling by quantitative phosphoproteomics

“…1G) 25,27 and in accordance with the preference of SCX chromatography and TiO 2 the majority of peptides contained only one phosphorylation (64%). 31,32 But a considerable proportion of double and multiphosphorylated peptides was also identified (Fig. 1H).…”

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

“…Titanium Dioxide Enrichment-TiO 2 enrichment of phosphopeptides was essentially performed as described (64). Briefly, 10 l of a 25% slurry of TiO 2 (MZ-Analysentechnik, Mainz, Germany) in 30 mg/ml 2,5-dihydroxybenzoic acid (Sigma Aldrich, St. Louis, MO) were added to each SCX fraction and incubated for at least 30 min at 4°C.…”

Enrichment Strategies for Identification and Characterization of Phosphoproteome

“…[53][54][55] To minimize these issues, loading and binding buffers during phosphopeptide enrichment with IMAC were often used to pH 2-2.5 containing with organic acid [e.g., trifluoroacetic acid (TFA), acetic acid, hydrochloric acid and formic acid]. 50 A variety of organic acids have been Immobilized metal ion affinity chromatography (IMAC) and (B) titanium dioxide (TiO 2 ) able for the specific enrichment of phosphopeptides from ordinary peptides used for high selectivity of IMAC, their influence resulted as follows: TFA > hydrochloric acid > formic acid > acetic acid. 53 On the contrary, the elution of phosphopeptides bound on IMAC was performed at strong basic conditions containing ammonium hydroxide, urea, or tris-buffer.…”