GL Sciences' Titansphere and MonoTip TiO 0 Phosphorylated Peptide / Phosphopeptide Purification and Enrichment Columns using TiO2, Titanium oxide, media


A completely new base material for HPLC column developed by GL Sciences consisting of spherical particles of titanium dioxide (TiO2). Titansphere has been found to be particularly effective in the selective enrichment of phosphorylated peptides from proteolytic digests. Titansphere offers greater selectivity for phosphopeptides than alternative methods, such as IMAC (immobilized metal affinity chromatography). Typically, IMAC shows significant binding of non-phosphorylated peptides which have multiple acidic residues. This problem is easily eliminated by using Titansphere. Typically, PEEK cartridges or non-metal capillaries containing Titansphere are used to selectively bind phosphorylated peptides from a peptide mixture. The phosphorylated peptides are then eluted from the column onto a reverse-phase HPLC column for further characterization or analyzed directly by MS.
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Figure 1 - Common column switching arrangement whereby phosphopeptides are captured on Titansphere precolumn and then eluted onto analytical RP column downstream before detection.
(click on image below to enlarge) Figure 2 - Detailed independent paper showing efficacy of Titansphere
(click on image below to enlarge) Figure 3 - Phosphopeptides extracted from tryptic digest of Casein using Titansphere TiO (A) Tryptic digest analyzed on Inertsil WP300 C8, 150 x 4.6mm column before using Titansphere; (B) Sample sample as in "A" after sample was run through Titansphere cartridge. Red arrows indicate phosphopeptides which were removed by Titansphere; (C) Phosphopeptides trapped on Titansphere cartridge and then eluted onto Inertsil WP300 C8 for analysis, showing specificity for phosphopeptides.

(click on image below to enlarge) Figure 4 - Peak shifts of tryptic digest of casein after treatment with alkaline phosphatase, indicating which peaks are potential phosphopeptides. Compare with Figure 3 above.

(click on image below to enlarge) Figure 5 - Peak shifts of phosphopeptides collected from tryptic digest of figure 3 and 4 after treatment with with alkaline phosphatase, indicating which peaks are likely dephosphorylated peptides.

Additional References shown below. A listing of PDF documents from ASMS (American Society of Mass Spectrometry) relating to TiO2 use for phosphopeptide enrichment can be found here. Phosphopeptide extraction Online, For digest of purified protein ● A. Schlosser, J. T. Vanselow, and A. Kramer, “ Mapping of phosphorylation sites by a multi-protease approach with specific phosphopeptide enrichment and NanoLC-MS/MS analysis ”, Analytical Chemistry 2005 Aug 15;77(16):5243-50. ● M. W. H. Pinkse, P. M. Uitto, M. J. Hilhorst, B. Ooms, and A. J. R. Heck, “ Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns ”, Analytical Chemistry 2004 Jul 15;76(14):3935-43. ● I. Kuroda, Y. Shintani, M. Motokawa, S. Abe, and M, Furuno, “ Phosphopeptide-selective Column-switching RP-HPLC with a Titania Precolumn ”, Analytical Sciences 2004 Sep;20(9):1313-9. PDF Available Here ●I. Kuroda, M. Motokawa, T. Fukaya, and M, Takahashi, “ Selective Detection of Phosphopeptides by Using 2-dimentional Capillary HPLC/MS ”, Poster made in 2004 for external release by GL Sciences inc. PDF. file is available. Offline, For digest of protein mixture ● M. R. Larsen, T. E. Thingholm, O. N. Jensen, P. Roepstorff, and T. J. D. Jorgensen, “ Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns ”, Molecular & Cellular Proteomics 2005; 4: 873-886. ● Yasuhiro Hirano, Masashi Segawa, Fumiko S. Ouchi¶, Yoshio Yamakawa¶, Kazuhiro Furukawa, Kunio Takeyasu, and Tsuneyoshi Horigome1 *Dissociation of Emerin from Barrier-to-autointegration Factor Is Regulated through Mitotic Phosphorylation of Emerin in a Xenopus* Egg Cell-free System*^ J. Biol. Chem., Vol. 280, Issue 48, 39925-39933, December 2, 2005			Phosphosugar extraction Online, For phosphosugars in extract of plant ● Y. Sekiguchi, N. Mitsuhashi, Y. Inoue, H. Yagisawa, and T. Mimura, “ Analysis of sugar phosphates in plants by ion chromatography on a titanium dioxide column with pulsed amperometric detection ”, Journal of Chromatography A 2004 Jun 11;1039(1-2):71-6. ● Y. Sekiguchi, N. Mitsuhashi, and T. Mimura, “ Sugar Phosphates Changes in Arabidopsis in Response to Phosphate Nutrition Measured by Improved Ion Chromatography with Pulsed Amperometric Detection Combined with a Titanium Dioxide Column ”, Plant Biotechnology 2004 21(2):143-150. Glyphosate extraction ● T. Ishiwata, K. Takai, H. Okada and K. Ohashi, “ ANALYSIS OF PHOSPHORUS-CONTAINING AMINO ACID-TYPE HERBICIDES AND THEIR METABOLITES IN HUMAN BLOOD BY EXTRACTION WITH A TITANSPHERE TiO COLUMN AND CAPILLARY ELECTROPHORESIS ”, Jpn J Forensic Toxicol, Vol 22, No.1, 2004:7-12. New assay method for ATP-dependant protein ● Y. Kimura, S. Shibasaki, K. Morisato, N. Ishizuka, H. Minakuchi, K. Nakanishi, M. Matsuo, T. Amachi, M. Ueda, and K. Ueda, “ Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column ”, Analytical Biochemistry 2004 Mar 15;326(2):262-6.