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Unique Polymer Technology (UPT)

For target ID without derivatization of the test molecule, choose Shantani's proprietary method, UPT.
[Low False Positive Identifications]


Working Principle

This method relies on a validated hypothesis that weak-molecular interaction forces of a drug-like molecule can be utilized in preparing their affinity matrix, without any derivatization of the compound. Though interactions are weak in nature, sum of multiple weak interactions will allow molecule to stay on a surface, that provide complementary interactions, for a time that is sufficient for it to further interact with its affinity protein partners in a biological sample. Thus prepared affinity matrix then can be used to enrich the target protein from biological sources.



Workflow

In UPT, the ‘bait-molecule’, without any chemical derivatization, is immobilized on proprietary polymers of Shantani. Shantani’s unique polymeric surface allows formation of complementary weak interaction bonds with ‘bait-molecule’ for it’s transient immobilization. Thus prepared ‘bait-molecule’ specific affinity matrix is then exposed to biological source, typically cell-lysates, for affinity capture of target protein. After appropriate incubation, polymer matrix is washed, and matrix bound proteins are isolated by dissolving the polymeric matrix in organic solvents. These proteins are then identified using mass-spectrometry and specific target proteins are de-convoluted by comparing the protein profile obtained from bait-molecule specific affinity matrix and multiple control experiments.



Key Advantages


Points of Consideration


References
Technical Note 1
(PDF, 103KB)
This note explains various steps involved in de-convoluting the right target(s) of small molecule using Shantani's UPT workflow.

Application Note 1
(PDF, 193KB)
This note describes the application of UPT in identifying/de-convoluting true positive targets of a non-derivatized ‘test’ molecule BIS-III.


FAQs
1. How “actually” molecules are getting immobilized on the polymer? Several unique chemical groups that can provide probability of pi-pi, cation-pi, hydrogen bonding and/or ionic interactions with organic molecules were incorporated in systematic pattern in the design of the polymer. Drug-like molecules in one or other orientation on polymer forms at least more than one kind of weak-interaction with specific chemical groups on polymer and thus get transiently immobilized on the polymer.


2. After molecules are immobilized over the polymer how it is made sure that these transient immobilization/interactions are not going-off during the washing, incubation and elution processes? How long the molecule stays on the polymer? In routine work-flow after molecules are immobilized on the polymer, extensive washing of polymer is carried out and small-molecules in the washes are quantified using LC-UV / LC-MS based method. The amount of molecule present in the washes direct correlated with the amount of molecule left bound to the polymeric surface. In typical experiment scenario 1-4 micromoles of molecule on an 8X8 cm polymeric surface stays for 30-60 minutes under constant orbital shaking at 20 RPM.


3. Are there more than one polymeric surface available? How many polymeric surfaces typically employed for the purpose? How polymeric surface is chosen? Based on solubility of the bait-molecule a few variations of polymeric surfaces are tested with a final goal that 1-4 micromoles of bait-molecule should remain retained on an 8X8 cm polymeric surface after 6 consecutive aqueous washing.


4. Whether method can be used for any given molecule? Typically following properties of the compound influence its immobilization on polymer in differentiation way (a) hydrophobic/hydrophilic nature (logP), (b) ionic bond formation capabilities and (c) molecular 3-D structure. Every molecule is unique and characterization of their retention behavior on the polymer is the only way forward. And hence before starting any target capture experiment retention of the molecule on the polymer is characterized. So practically any given molecule can be used for target capture experiment as long as they can be retained on the polymer for appropriate time.


5. How much molecule and cell-lysate will be needed for one target capture experiment? Typically 12-15 mg of molecule and 15-20 mg of proteins/cell lysate is sufficient.


6. What mass-spec instrument is used for peptide/protein identification purposes? Agilent 6540 UHD Accurate Mass Q-TOF is employed for the purpose of protein identification. In typical workflow using ProtID CHIP 150 II and Agilent CHIP cube interface tryptic peptides are gradually eluted and ionized and ionized peptides are then directly infused into the mass-spec for MS and MS/MS data acquisition. Peptide sequences are established through MS and MS/MS signatures and identified peptides are then searched against redundant protein databases to link it to a protein.


7. How typically specific protein targets are deconvoluted? Typically experiments are carried in duplicate or triplicate along with a control experiment (using polymeric surface where molecules are not immobilized). Protein identified from bait-molecule experiments are compared with the protein identified from the control. Proteins that are (a) exclusively identified in all the triplicate bait-molecule runs and/or (b) identified with significantly more number of peptides/higher signal intensity in bait-molecule run compared to control run are considered as deconvoluted targets.