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Frequently Asked Questions

1. What protocols does Shantani follow for protein identification using mass-spectrometry? Shantani follows the protocols published in Saxena et al, An Immuno-Chemo-Proteomics Method for Drug Target Deconvolution, J. Proteome Res. (2008) 7 pp. 3490-3497. In brief, eluted proteins are separated on the SDS-PAGE gel. Whole gel lane is cut as multiple gel-bands. Proteins in the bands are reduced, alkylated and then trypsin digested. Peptides are extracted from the bands and then infused into the mass-spectrometer for MS and MS/MS analysis.

2. 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.

3. How are specific protein targets usually deconvoluted?

For all target-enrichment methods (SCLS, UPT, TBB):
Typically experiments are carried in duplicate or triplicate along with appropriate control experiments. Protein identified from bait-molecule experiments are compared with the protein identified from the control. Proteins that are (a) exclusively identified in all the duplicate/triplicate bait-molecule runs and/or (b) identified with significantly more number of peptides/higher signal intensity in bait-molecule run compared to all the control run are considered as deconvoluted targets.

4. What types of control experiments does Shantani run in parallel to establish the specificity of the pull-downs? How is the specific target(s) information actually deconvoluted? Shantani runs one negative control for each experiment. In one experiment, the 'bait/test' molecule not coupled to the probes is utilized for pull-down experiments; in the other experiment, the cells or cell/lysates are pre-incubated with uncoupled 'bait/test' molecule and then the target pull-down is performed using the bait molecule coupled probes.

Specificity of the target capture is calculated based on the presence of particular protein in actual experiment over its presence in all the three experiments. We are also building a robust label-free quantification approach to increase the confidence in our deconvolution studies.

5. How much cell-lysate is needed? Can Shantani culture the cells? We prefer that ~15 mg of protein(cell-lysate) is provided for the purpose. In case not, we can culture the cells and generate the required amount of cell-lysate as long as either seed-culture of the cell and culture protocol is provided OR commercial source for the same is located.

6. Will Shantani share with us the .raw files of mass-spec data? Shantani performs a robust analysis of mass-spec data to obtain the list of deconvoluted proteins and usually does not share the .raw file data. However, if you have a critical need, we will share the data.

7. For how long will Shantani support the data? Shantani provides data support for 12 months, at no additional cost, from the date of project completion. Beyond that period, we can maintain the data at an additional cost to client.

8. What is the typical affinity of the target that can be identified?

False Positive Rate (FPR): The total number of de-convoluted proteins as targets minus the actual number of targets (usually confirmed during validation) over total number of de-convoluted targets.

Typical Affinity (Kd) of Identified Targets with bait-molecule: Bait-molecule-Target Interaction studies were carried out after target identification experiments to understand the advantages and limitations of different methodologies.

Subcellular Location Specific~ 20%Kd < 1 µm
Unique Polymer Technology~ 40%Kd < 1 µm
Traditional Bead & Biotinylation~ 40%ND
Computational Docking~ 80%Not Applicable (only predictive Kd)