FAQ – Frequently Asked Questions

1) When you perform LRC-TriCEPS for target identification are cells alive?

Yes, the cells are alive under near physiological conditions.

2) Do you need to genetically manipulate the cells?

No genetic manipulation (eg. ectopic expression) is needed.

3) Can you work with stimulated cells (i.e. activated T-Cells)?

Yes, in fact we encourage to find the best growth condition for the cells of your interest.

4) I have developed a complex way of growing cells to respond to my ligand of interest. Can I perform an LRC-TriCEPS experiment?

When cell culturing is not easily transferable, Dualsystems provides the TriCEPS material and the customer performs ligand coupling, receptor capture and collection of cells. The frozen cell pellets are then shipped to Dualsystems for further processing.

5) I do not know which cell type / cell line expresses the unknown target(s) of my ligand. Can you help?

Yes, with the Flow-TriCEPS kit you can couple your ligand of interest to a TriCEPS Version that contains a biotin. The biotin can then be used in flow cytometry to visualize the binding of your ligand to the cells. You can use the system to screen different cell types/ cell lines for the expression of the unknown target(s).

6) How much material do I need from my ligand of interest to perform one LRC-TriCEPS experiment?

In average, you need 300microg for a standard experiment using protein/antibodies as ligand. In case you have limited amounts of ligand available, we could design a customized experiment for you and then 100microg might also be enough. For peptides and small molecules, drop us a line.
7) How many cells are needed for one LRC-TriCEPS experiment?

The exact number of cells needed for one LRC experiment depends on the cell line. As a rule of thumb, a cell pellet with a volume of 50-100 ul is needed per sample. For most adherent cell lines that corresponds to 10-20×106   cells per sample. In a typical LRC-TriCEPS experiment, at least two treatment arms are performed in parallel: one with the ligand of interest and a second with a control ligand (that is, a ligand with a known target). Both arms are performed in triplicates to allow statistical analysis therefore roughly 60-120×106 cells in total are required.

In the case of primary cells from humans or animals (i.e. primary T cells) where the number of cells may be a limiting factor, we recommend performing the treatment arms in singletons but analyzing at least 3 different donors.

8) Can I use primary cells (i.e.T-cells) for my LRC-TriCEPS experiment?

Yes, from one donor it is expected to receive enough cells to perform an experiment with the ligand of interest and one control. As the number of cells per T-cell donor is not very large, the experiment is performed in singletons and triplicates are performed on the level of LC-MS/MS measurements. We recommend to perform an experiment on at least 3 different donors to see donor to donor variation.

9) I have adherent cells, is it possible to perform an LRC-TriCEPS experiment?

Yes, you can either detach the cells by adding EDTA or scraping prior to treatment with TriCEPS coupled ligand. However, we advise to add the TriCEPS coupled ligands directly to the plate so that the expression pattern of the cells is not altered during detachment of cells.

10) How large is TriCEPS?

Ca. 1.2 kDa.

11) Can I adapt the LRC-TriCEPS experiment so that I have the optimal binding conditions for my ligand of interest?

To find the best condition we advise to use Flow-TriCEPS where you can visualize the binding of your ligand to the cells in flow cytometry (available as kit or service). It is advised to perform optimization experiments to find the best binding conditions and then use those conditions during receptor capture in the main LRC-TriCEPS experiment. Receptor capture during the standard LRC-TriCEPS workflow is performed at pH 6.5 for 90min at 4oC. During a Flow-TriCEPS experiment, the effect of at least three factors on ligand binding is evaluated: pH, time and temperature. Additional factors (such as presence of a co-factor) can be also investigated.

12) Is my ligand still functional active/ able to bind to its target when coupled to TriCEPS?

If you have a functional assay we advise to couple TriCEPS to your ligand of interest and test if it is still functionally active compared to not coupled-ligand. We provide the Flow-TriCEPS kit that contains a biotinylated TriCEPS version where you can couple six times 20microg of ligand and visualize if the coupling worked by dot blot.

13) Where does TriCEPS couple to the ligand of interest?

To all primary amines. In peptides and proteins mainly to the N-Term and the Lysines. If you have a small molecule you might need to produce a derivative that contains a primary amine.

14) I expect that my ligand binds to more than one target. Can the LRC-TriCEPS technology identify multiple targets?
Yes, targets and off-targets can be identified in the same experiment.

15) I expect that my ligand binds to a receptor that forms homo or heterodimers. Can the LRC-TriCEPS technology identify these receptors?

Yes, since the cells are still alive during ligand-target interaction and the targets are in their natural micro-environment receptors are able to form dimers.

16) I have no expectation what the target of my ligand is. Can the LRC-TriCEPS technology still help to identify the unknown targets of my ligand?
Yes, we only need cells that express the unknown target. (see flow TriCEPS for cell type identification)

17) I suspect my ligand binds first an extracellular matrix protein that then binds to a protein to transduce the signal intracellularly. Can the LRC-TriCEPS technology still help to identify the unknown targets of my ligand?

Yes, we have shown that we also can identify co-receptors.

18) Are there any limitations concerning the targets that can be identified?

The target has to be a protein with at least one glycosylation site. It is expected that more than 90% of the cell membrane associated proteins are glycosylated.

19) How long does an LRC-TriCEPS experiment take?

From the moment the TriCEPS coupled ligands can be added to the cells it takes 4-5weeks till the report is provided.

20) How many target candidates are expected to be identified?

A candidate list of <5 is expected.

21) Do I need to artificially overexpress potential target proteins to be able to show the binding?

No, cells are not genetically manipulated.

22) Is there a benefit if I perform an experiment for my ligand of interest with more than one control?

Yes, in the LRC-TriCEPS technology we do relative quantification of the surface proteins. We always do a pairwise comparison (ligand of interest compared to control 1, ligand of interest compared to control 2 etc.) If more than one control is used better evaluation of data can be performed.

23) Is there a benefit if I perform an experiment for my ligand of interest on different cell types?

Yes, it is interesting to see whether your ligand binds to the same targets across cell lines or different ones depending on the cell type.

24) On what type of cells can you perform an LRC-TriCEPS experiment to identify the targets?

Any cell type that has glycosylation machinery.

25) In what kind of buffer should the ligand of interest be available for use in a LRC-TriCEPS experiment?

Best is HEPES pH 8.2 or PBS pH 7.4

Most important is that the buffer does not contain any primary amines. Therefore Tris-HCl cannot be used as a buffer for the ligands unless removed (i.e. dialysis).

26) Can you guarantee the success of the LRC-TriCEPS experiment?

We can cover the technical risk. If the positive control (i.e. transferrin, anti-CD28 antibody, anti-EGFR antibody) does not lead to identification of the known targets, we repeat the experiment once for free.

27) What is the number of cells needed for the pre-experiment using flow cytometry to identify the best binding conditions?

Normally ca. 5×106 cells are enough for a pretest.

28) Can I use the LRC-TriCEPS technology to identify the mode of action of my ligand of interest?

Yes, we can identify target receptors or co-receptors and in some cases also part of a protein complex that binds to the ligand of interest and therefore help to identify the mode-of-action of a molecule.

30) Can the LRC-TriCEPS technology be used for target deconvolution studies?

Yes, we can identify the target of your drug on living cells.

31) I want to do cell-cell interaction studies. That means my ligand of interest is a membrane protein and I suspect that its target is also a cell membrane protein on another cell. Can you help?

Yes, we can use the extracellular domain of your ligand of interest with i.e. Fc-fused and identify the binding partner on another cell.

32) I have a ligand with a low affinity to its targets. What is the sensitivity of the LRC-TriCEPS technology?

This is different for each ligand-receptor pair. In one case, we identified targets that bind the ligand with 2.5microM binding affinity. Furthermore, we were able to identify a receptor that was expressed with less than 2000 molecules per cells with a ligand with less than 1microM binding affinity.

33) Should the ligand be purified?

The use of a pure ligand depleted of unknown contaminants is highly recommended.

34) What is the concentration of the ligand used during an LRC-TriCEPS experiment?

During a typical LRC-TriCEPS experiment the ligand of interest is added on the cells during receptor capture at a concentration of 6 ug/ml.

35) Why do I need a control?

In a typical LRC-TriCEPS experiment, at least two treatment arms are performed in parallel: one with the ligand of interest and a second with a control ligand (that is, a ligand with a known target). The first step of the LRC includes the conjugation of the two ligands to TriCEPS. The TriCEPS-ligand conjugates are then incubated with previously oxidized cells under near-physiological conditions. During this phase, transient and stable ligand-receptor interactions will result in covalent capture events between TRICEPS and nearby carbohydrates. After the receptor-capture reaction, the cells are lysed, proteins are isolated and processed for MS-based analysis. Upon identification, the relative abundance of cell surface proteins in the ligand samples are compared to those in the control sample using MS1-based label-free quantification. Randomly identified cell surface proteins are expected to have equal abundance in both samples, whereas the corresponding receptors are found enriched in the ligand sample

36) What type of information I will receive upon completion of the experiment?

Upon completion of the LRC-TriCEPS experiment, the following items will be provided to you.

1) A detailed report outlining
a) experimental design and procedures
b) results in a format of figures and tables
c) information regarding identified candidates
d) detailed discussion on the overview of the experiment

2) A list of membrane associated proteins identified during the LRC-TriCEPS experiment along with information regarding the differential abundance across tested conditions.

3) Raw files of the LC-MS/MS runs (upon request).

37) How stable is the interaction between the ligand and the target?

Transient and stable ligand-receptor interactions result in covalent capture events between TRICEPS and nearby carbohydrates.

38) Can my ligand be bound in beads or plate?

No, the ligand cannot be immobilized.

39) My ligand has a tag. Is that a problem?

No, the existence of a tag does not pose a problem.