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Metal Conjugated Tetramers

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CRStevens

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Post Thu Apr 12, 2018 2:11 pm

Metal Conjugated Tetramers

Hi All,

I know Fluidigm keeps talking about metal conjugated tetramers and that someday they will release this product, but has anyone cooked up a homegrown solution to this question? I am assuming the issues are with the chemistry of linking the polymer to a Neutravidin molecule? Has anyone tried to use an amine binding linker and conjugate the X8 polymer to the linker?

Any help from the group would be much appreciated.

-Chad
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mleipold

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Location: Stanford HIMC, CA, USA

Post Thu Apr 12, 2018 2:50 pm

Re: Metal Conjugated Tetramers

Hi Chad,

A while back, I did come up with a way to conjugate MAXPAR polymer to streptavidin or anything else that lacks Cys, or where the thiol-chemistry would screw up the biomolecule (like some of the lectins I published on with Mark NItz). I have attached the protocol: basically, reverse-engineering a thiol-terminated polymer and using Sulfo-SMCC on the biomolecule to attach it. We tested it with some biotin-labeled flow antibodies, and it worked.

The only caveat: I've heard that the biotin-binding pocket of streptavidin monomers contains a Lys which can be derivatized by small molecules (such as Sulfo-SMCC). For a biotin-targeting secondary method like I tested, it's not critical to maintain all 4 biotin-binding sites in a streptavidin tetramer. However, for use in tetramer staining, you'd need to keep all of them for avidity interactions. Evan Newell told me this was one reason why he engineered the mono (or poly) Cys into the mutant streptavidins he made for the CyTOF tetramers.

So, give it a try, but with careful appropriate control experiments. Please report back whether it worked!


Mike
Attachments
MAXPAR-DTT-SSMCC-amine protocol.docx
(116.02 KiB) Downloaded 455 times
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mleipold

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Post Thu Apr 12, 2018 3:10 pm

Re: Metal Conjugated Tetramers

Here's the paper where we developed the Sulfo-SMCC protocol for lectins: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646003/

The main reason I'm mentioning it is that you can use MALDI to "count" the number of Sulfo-SMCC attached to your biomolecule. In my experience, the MALDI process (matrix addition through laser shot) usually dissociates multimers into monomers, so you'd have to back-calculate for the number of Sulfo-SMCC per multimer. But that would give you an upper limit to the number of MAXPAR that could be attached.

In my experience with the lectins: the number of Sulfo-SMCC attached can vary significantly between biomolecules, but the number attached to a *given* biomolecule is reproducible from batch to batch. However, I didn't check the streptavidin by MALDI to be able to give you a number.
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rakondy

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Posts: 2

Joined: Mon Jan 18, 2016 6:24 pm

Post Mon May 07, 2018 6:05 pm

Re: Metal Conjugated Tetramers

Hi Chad ,
Were you able to use the thiol-terminated polymer + Sulfo-SMCC streptavidin for successful tetramer staining? Pearce sells a maleimide activated streptavidin and it will be easier for me to do this than try and make the 6-cysteine version of streptavidin.

Thank you
Rama
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mleipold

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Post Mon May 07, 2018 6:21 pm

Re: Metal Conjugated Tetramers

Hi Rama,

To confirm: this is the maleimide-activated streptavidin you're referring to?

ThermoScientific, Pierce: Catalog number: 21102
https://www.thermofisher.com/order/cata ... -srp-21102


Mike
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rakondy

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Post Mon May 07, 2018 6:23 pm

Re: Metal Conjugated Tetramers

Thank you for responding. Yes it is. I emailed techsupport to enquire in case there is a possibility that the biotin binding sites remain intact.

UPDATE: The company responded promptly, it doesn't look like they have data on the biotin binding sites (for the maleimide activated streptavidin) but the tech support person did not recommend using it for tetramers.
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jul 18, 2018 5:41 pm

Re: Metal Conjugated Tetramers

Hi Chad,

I stumbled upon this paper today:
"Tuning the Diels–Alder Reaction for Bioconjugation to Maleimide Drug-Linkers"
DOI: 10.1021/acs.bioconjchem.8b00320

Here, the authors use an NHS-ester small molecule to install various dienes onto the antibody scaffold (amine chemistry, since it's NHS-ester). They then react that with a maleimide-containing drug adduct to form the Diels-Alder product.

If you could find/make an appropriate NHS-ester diene, this could be another way to install MAXPAR.


Mike
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XMUCyTOF

Participant

Posts: 9

Joined: Tue Aug 07, 2018 3:09 am

Post Mon Jun 14, 2021 3:21 am

Re: Metal Conjugated Tetramers

rakondy wrote:Thank you for responding. Yes it is. I emailed techsupport to enquire in case there is a possibility that the biotin binding sites remain intact.

UPDATE: The company responded promptly, it doesn't look like they have data on the biotin binding sites (for the maleimide activated streptavidin) but the tech support person did not recommend using it for tetramers.


Hi Rama, Were you able to use the (ThermoScientific, Pierce: Catalog number: 21102) streptavidin for successful tetramer staining?
Could you share me some experience? I also try to make the 6-cysteine version of streptavidin but it's not easy.
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hwtchen

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Joined: Thu May 07, 2015 6:35 pm

Location: Sickkids, Toronto, Canada

Post Tue Jun 29, 2021 6:58 pm

Re: Metal Conjugated Tetramers

Hi Mike,
I have a question about your MAXPAR-DTT-SSMCC-amine protocol. Can you clarify what is the volume of the polymer solution when adding lanthanide and TCEP at the metal loading step? I might not be spinning long enough to concentrate.
Thanks.

Tina

mleipold wrote:Hi Chad,

A while back, I did come up with a way to conjugate MAXPAR polymer to streptavidin or anything else that lacks Cys, or where the thiol-chemistry would screw up the biomolecule (like some of the lectins I published on with Mark NItz). I have attached the protocol: basically, reverse-engineering a thiol-terminated polymer and using Sulfo-SMCC on the biomolecule to attach it. We tested it with some biotin-labeled flow antibodies, and it worked.

The only caveat: I've heard that the biotin-binding pocket of streptavidin monomers contains a Lys which can be derivatized by small molecules (such as Sulfo-SMCC). For a biotin-targeting secondary method like I tested, it's not critical to maintain all 4 biotin-binding sites in a streptavidin tetramer. However, for use in tetramer staining, you'd need to keep all of them for avidity interactions. Evan Newell told me this was one reason why he engineered the mono (or poly) Cys into the mutant streptavidins he made for the CyTOF tetramers.

So, give it a try, but with careful appropriate control experiments. Please report back whether it worked!


Mike
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Jun 29, 2021 7:14 pm

Re: Metal Conjugated Tetramers

Hi Tina,

To confirm: you're asking about finishing step A2 and moving into step A3? If so, this would be 95uL from the L buffer added at the end of Step A2.

BTW, Part A and Part B should be performed in parallel like in the standard MAXPAR protocol. Once you have reduced the potential disulfide with the TCEP in Step A3 and washed in A4, you should move directly on to part C/conjugation to make sure the disulfides don't re-oxidize (-S back to -S-S-).


Mike
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