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


GL Sciences has introduced a highly selective new kit for phosphopeptide isolation from tryptic and other proteolytic digests. Unlike its predecessor, MonoTip TiO which was operated by pipette, Phos-TiO columns are centrifuge spin columns, available in 1mg/10uL and 3mg/200uL sizes, in packages of 24 or 96. Adaptors are available (as they are reusable, these must be ordered separately from the kits) to spin the columns individually or as a 96 well plate. In addition to the spin columns, the kits includes washing and elution buffers and collection and waste tubes. The unique processing of the titanium dioxide based media synthesized by GL Sciences shows unparalleled selectivity for phosphopeptides, with virtually zero non-specific binding of non-phosphorylated peptides under the specified operating conditions. There are only 4 steps in the enrichment process and total processing time is typically under 40 minutes.
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Standardized Procedure Not surprisingly, the enrichment process consists of conditioning the Phos-TiO spin columns, applying the sample under conditions promoting binding of phosphopeptides to the media, rinsing non-bound molecules off of the columns, and then eluting the purified phosphopeptides, as indicated in the schematic below: Each step is performed using a centrifuge, and Phos-TiO kits require reusable adaptors used to secure the spin columns to either individual collection tubes or to a 96-Well Plate. As these items are reusable, they are sold and must be ordered (only once) separately from the Phos-TiO kits. The centrifuge adaptors for individual tubes are the same for both the 10uL and 200uL spin columns. Separate 96-well plate adaptors are sold for 10uL and 200uL tips, as shown below: Adaptors for individual collections tubes are shown at right. These are used for both 10uL and 200uL Phos-TiO spin columns and are reusable. The order number for a package of 24 adaptors is 140001.
Adaptors for 96-Well plate processing are available for both the 10uL spin columns and 200uL spin columns. These are sold individually and are reusable. Order No. for 10uL 96-well plate adaptor is 140002 and that for the 200uL 96-well plate adaptor is 140003.
Phos-TiO kits are extremely selective for phosphopeptides The mass spectra data below shows a comparison of Phos-TiO versus alternative titanium based products as well as IMAC techniques. Phos-TiO shows the best sensitivity, and shows superior enrichment of multi-phosphorylated peptides (4+) compared to IMAC. In this example, enrichment efficiency of phosphopeptides from a tryptic digest of 2.5 �g β-casein using MALDI-TOF/MS.
The following graph shows recovery and selectivity among four commercially available phosphopeptide enrichment kits in addition to Phos-TiO. Phos-TiO shows virtually no non-specific adsorption and provides the best results overall: The chart below compares Phos-TiO and IMAC techniques in isolating phosphopeptides from a tryptic digest of 100ug of a HeLa cell lysate. Note the superior selectivity and recovery provided by Phos-TiO:
References: (1) Optimizing a selective enrichment conditions for phosphopeptides from tryptic digest of peptides & a quality comparison of titanium dioxide based columns and IMAC column. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Molecular & Cellular Proteomics 2005; 4: 873-886. Larsen, et. al. (2) Verifying the dynamic of phosphorylation strength and time lapse of EGF-stimulated intracellular signaling factor (ex: GTPase, transcription factor, kinase..etc) using titanium dioxide. Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks. Cell 127, 635-648, November 3, 2006, Olsen, et. al. (3) Verifying a highly selective purification & enrichment method for phosphopeptides from tryptic digests of Hela cell lysate. Phosphopeptide Enrichment by Aliphatic Hydroxy Acid-Modified Metal Oxide Chromatography for NanoLC-MS/MS in Proteomics Applications. Molecular & Cellular Proteomics 2007; 6: 1103 - 1109. Sugiyama, et. al. (4) The influence of samples containing a surface active agent or denaturing agent (ex: SDS, urea, etc.) using titanium dioxide for phosphopeptide enrichment. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun. Mass Spectrom. 2007; 21: 3635 - 3645