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539722 ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit

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      Overview

      Replacement Information
      Description
      OverviewThe ProteoExtract® Phosphopeptide TiO2 Enrichment Kit is a useful tool for enrichment of phosphorylated peptides from complex samples by a highly selective Titanium oxide solid phase. It enhances the ability to routinely identify and characterize large numbers of phosphorylated species within complex protein mixtures taking advantage of a novel titanium dioxide material. Captured and eluted phosphopeptide fractions are ready for MALDI- and ESI- mass spectrometry analysis.
      Catalogue Number539722
      Brand Family Calbiochem®
      Application Data
      Titanium dioxide allows for selective and sensitive phosphopeptide enrichment with low background from complex mixtures. A complex peptide mixture derived from a tryptic digest of porcine liver extract was spiked with α-casein and two synthetic phosphopeptides (see below for concentrations). The samples were subsequently processed using the ProteoExtract® Phosphopeptide Enrichment TiO2 Kit as outlined in the Detailed Protocol. Mass spectrometry analysis was performed using ESI-LC/MS equipment operated in positive mode. (A) Unprocessed sample (B) Phosphopeptides recovered using the ProteoExtract® Phosphopeptide Enrichment TiO2 Kit. Spiked Phosphopeptides: 1. 50 nM Angiotensin ~6 pmol/125 µl; ~7 ng/125 µl 2. 67 nM Calcineurin Substrate ~8 pmol/125 µl; ~18 ng/125 µl 3. 140 nM α-Casein ~18 pmol/125 µl; ~400 ng/125 µl
      Materials Required but Not Delivered• Micropipettes and tips; 10 µl, 200 µl, and 1 ml
      • Microcentrifuge and rotor for >10,000 x g
      • 1.5 ml microcentrifuge tubes
      References
      Product Information
      Detection methodMass Spectrometry
      Form100 Isolations
      FormatPhosphopeptide enrichment
      Kit containsTiO₂ Phosphobind Resin, 10X TiO₂ Phosphobind Buffer, Wash Buffer 1, Wash Buffer 2, TiO₂ Elution Buffer, Dihydroxybenzoic Acid, and a user protocol.
      Applications
      Biological Information
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit is designed to enrich phosphorylated peptides from complex samples using a solid phase, highly selective, titanium oxide. The enriched phosphopeptide fraction is directly compatible with downstream analysis by MALDI and ESI mass spectrometry.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C. Following reconstitution of the Dihydroxybenzoic Acid refrigerate (4°C) for short-term storage or aliquot and freeze (-20°C) for long-term storage; reconstituted Dihydroxybenzoid Acid is stable for up to 6 weeks at 4°C or for up to 6 months at -20°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsTiO₂ Phosphobind Resin, 10X TiO₂ Phosphobind Buffer, Wash Buffer 1, Wash Buffer 2, TiO₂ Elution Buffer, Dihydroxybenzoic Acid, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      539722 0

      Documentation

      ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit SDS

      Title

      Safety Data Sheet (SDS) 

      ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit Certificates of Analysis

      TitleLot Number
      539722
      User Protocol

      Revision18-September-2007 JSW
      Form100 Isolations
      FormatPhosphopeptide enrichment
      Detection methodMass Spectrometry
      StorageUpon arrival store the entire contents of the kit at 4°C. Following reconstitution of the Dihydroxybenzoic Acid refrigerate (4°C) for short-term storage or aliquot and freeze (-20°C) for long-term storage; reconstituted Dihydroxybenzoid Acid is stable for up to 6 weeks at 4°C or for up to 6 months at -20°C.
      Intended useThe ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit is designed to enrich phosphorylated peptides from complex samples using a solid phase, highly selective, titanium oxide. The enriched phosphopeptide fraction is directly compatible with downstream analysis by MALDI and ESI mass spectrometry.
      BackgroundIn eukaryotic cells, post-translational modifications of proteins such as phosphorylation and dephosphorylation are involved in numerous metabolic pathways and in the transmission of signals that control proliferation, differentiation, and apoptosis. Deregulation of the tightly controlled balance between phosphorylation and dephosphorylation may lead to serious pathological conditions. Determining the site(s) of these regulatory phosphorylations is therefore important for understanding essential signaling pathways and for gaining insight into the molecular basis of diseases. The identification of phosphorylation sites is routinely accomplished by mass spectrometry (MS). Due to the high complexity of cellular proteome fractions there is a general need for specific and efficient strategies for enrichment of phosphorylated peptides prior to MS. Efficient enrichment strategies help to compensate for the low stoichiometry of phosphopeptides relative to their unphosphorylated counterparts and for poor ionization and ion suppression effects inherent to MS analysis.
      Principles of the assayThe ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit uses a novel titanium dioxide material to enable identification of large numbers of phosphorylated species from complex protein mixtures. The titanium dioxide is highly selective for phosphorylated peptides in the presence of abundant non-phosphorylated peptides. The protocol and buffers are optimized to produce high yields of the phosphopeptides. Enrichment and selectivity for phosphopeptides is further improved by using a 2,5-DHB "displacer" concentration that is directly compatible with LC-MS and MALDI-MS analysis.
      Materials provided• TiO₂ Phosphobind Resin (Kit Component No. KP31796): 1 vial, 6 ml TiO₂ suspension
      • TiO₂ Phosphobind Buffer (Kit Component No. KP31795): 1 vial, 20 ml
      • Wash Buffer 1 (Kit Component No. KP31491): 1 vial, 20 ml
      • Wash Buffer 2 (Kit Component No. KP31492): 1 vial, 20 ml
      • Elution Buffer (Kit Component No. KP31493): 1 vial, 3 ml
      • Dihydroxybenzoic Acid (Kit Component No. KP31797): 1 vial, 2 g powder
      Materials Required but not provided• Micropipettes and tips; 10 µl, 200 µl, and 1 ml
      • Microcentrifuge and rotor for >10,000 x g
      • 1.5 ml microcentrifuge tubes
      Detailed protocolNote: Warm all reagents to room temperature. Prior to starting the assay weigh 1 g Dihydroxybenzoic Acid and add it to the vial of TiO2 Phosphobind Buffer and mix well. Dilute trypsin-digested samples at least 1:4 with TiO2 Phosphobind Buffer containing Dihydroxybenzoic Acid (e.g., 50 µl sample + 150 µl TiO2 Phosphobind Buffer) to achieve a final volume of 100-200 µl (samples that have been digested with trypsin usually have a basic pH).

      1. Mix the TiO2 Phosphobind Resin by vortexing until completely suspended.
      2. Transfer 50 µl of the homogeneous suspension to a microcentrifuge tube, centrifuge the tube for 3 min at 2000–2500 x g. Completely remove the supernatant and discard.
      3. Add the diluted sample to the TiO2 Phosphobind Resin, mix carefully, and incubate with gentle agitation for 10 min at room temperature. The use of a mixer at 1100 rpm is recommended. Alternatively, vortex the tube briefly several times during the incubation time.
      4. Centrifuge the tube for 3 min at 2000–2500 x g, remove the supernatant completely, and discard.
      5. Add 100 µl Wash Buffer 1 to the tube, mix carefully, and centrifuge for 3 min at 2000–2500 x g. Remove the supernatant completely and discard. Repeat for a total of 2 washes.
      6. Add 100 µl Wash Buffer 2 to the tube, mix carefully, and centrifuge for 3 min at 2000–2500 x g. Completely remove the supernatant and discard the supernatant. Repeat for a total of 2 washes.
      7. After removing the supernatant in the final wash in step 6, centrifuge the tube for 1 min at 2000–2500 x g and completely remove the remaining supernatant.
      8. Add 30 µl Elution Buffer to the tube, mix carefully by pipetting up and down, and incubate with gentle agitation for 10 min at room temperature. The use of a mixer at 1100 rpm is recommended. Alternatively, vortex the tube briefly several times during the incubation time.
      9. Centrifuge the tube for 5 min at 10000 x g and carefully transfer the supernatant to a new microcentrifuge tube. Avoid transferring the TiO2 Phosphobind Resin.
      10. Centrifuge the tube containing the supernatant (step 9) for 3 min at 10000 x g. Collect the supernatant in a new tube and save it for further analysis of the captured and purified phosphopeptides. Avoid transferring the TiO2 Phosphobind Resin; if necessary leave a small volume of supernatant in the tube to avoid transferring the TiO2 Phosphobind Resin.
      11. In the event that some TiO2 Phosphobind Resin is transferred, repeat step 10.
      Assay characteristics and examplesThe data in Figure 1 demonstrates that phosphopeptide capture is efficient and specific using the ProteoExtract® Phosphopeptide Enrichment TiO₂ Kit. The predominant signals are derived from phosphopeptide ions (marked with arrows) and the majority of non-phosphorylated peptides are completely removed (low background). Only monophosphorylated phosphopeptides are detectable under these conditions.


      Figure 1: Phosphopeptide Enrichment

      Titanium dioxide allows for selective and sensitive phosphopeptide enrichment with low background from complex mixtures. A complex peptide mixture derived from a tryptic digest of porcine liver extract was spiked with α-casein and two synthetic phosphopeptides (see below for concentrations). The samples were subsequently processed using the ProteoExtract® Phosphopeptide Enrichment TiO2 Kit as outlined in the Detailed Protocol. Mass spectrometry analysis was performed using ESI-LC/MS equipment operated in positive mode. (A) Unprocessed sample (B) Phosphopeptides recovered using the ProteoExtract® Phosphopeptide Enrichment TiO2 Kit. Spiked Phosphopeptides: 1. 50 nM Angiotensin ~6 pmol/125 µl; ~7 ng/125 µl 2. 67 nM Calcineurin Substrate ~8 pmol/125 µl; ~18 ng/125 µl 3. 140 nM α-Casein ~18 pmol/125 µl; ~400 ng/125 µl

      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      ProteoExtract® is a registered trademark of Merck KGaA, Darmstadt, Germany
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.