Optimizing TiO₂-Based Phosphopeptide Enrichment for Automated Multidimensional Liquid Chromatography Coupled to Tandem Mass Spectrometry

Abstract

An automated online multidimensional liquid chromatography system coupled to ESI-based tandem mass spectrometry was used to assess the effectiveness of TiO 2 in the enrichment of phosphopeptides from tryptic digests of protein mixtures. By monitoring the enrichment of phosphopeptides, an optimized set of loading, wash, and elution conditions were realized for TiO 2 . A comparison of TiO 2 with other resins used for phosphopeptide enrichment, Fe(III)-IMAC and ZrO 2 , was also carried out using tryptic digests of both simple and moderately complex protein mixtures; where TiO 2 was shown to be superior in performance.

Automated Phosphoproteome Analysis for Cultured Cancer Cells by Two-Dimensional NanoLC-MS Using a Calcined Titania/C18 Biphasic Column

“…Winkler and Marme reported that another commercial titania (Sachtopore from Sachtleben Chemie GmbH) was also anatase form. 34 In this study, we chose Titansphere based on the results of a comparison between these commercial beads, 29 and prepared rutile beads by the calcination method according to Tani et al 35 By heating Titansphere at 800˚C for 2 h, we successfully obtained rutile beads, as shown in Fig. 1B.…”

“…17,19,24,28,29 However, no system has yet been applied to real complex samples, such as cell lysates, although recently Cantin et al analyzed a moderately complex sample with a titania column-trap column system. 29 The reason for the failure to achieve practical application is mainly that the dynamic range between phosphorylated and non-phosphorylated peptides is much wider in real samples, and this results in poor identification of phosphopeptides.…”

Large‐scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography

“…The first one is chemical reaction-based methods which include beta-elimination of the phosphate group and subsequent introduction of an affinity tag [11], and then captured by beads and released [12]. The second one is chromatography based methods which include affinity chromatography with immobilized metal ions (IMAC) [13][14][15][16][17][18][19][20][21][22][23], metal oxides such as titanium dioxide (TiO 2 ) and zirconium dioxide (ZrO 2 ) [24][25][26][27][28][29][30][31], and strong cation exchange chromatography (SCX) [32][33][34][35]. Among all of these methods, IMAC with Fe 31 is the most popular technique for the enrichment of phosphorylated peptides.…”

Linking the proteins—Elucidation of proteome‐scale networks using mass spectrometry

“…As an alternative, phosphoramidate chemistry has been developed to allow coupling phosphopeptides to a dendrimer (Tao et al, 2005;Bodenmiller et al, 2007c). The different enrichment strategies, IMAC and TiO 2 (Cantin et al, 2007;Liang et al, 2007;Marcantonio et al, 2008;Wilson-Grady, Villen, & Gygi, 2008), as well as phosphoramidate chemistry (Bodenmiller et al, 2007b), provide distinct but overlapping sets of phosphopeptides. Phosphotyrosine-containing peptides could be handled differently and targeted more specifically: the availability of highly specific antibodies that recognize this phosphomotif allowed the large-scale affinity purification of pTyrcontaining proteins/peptides.…”