Try to do On-bead digestion. Thats it!

On-bead digestion doesn't work well because the streptavidin gets digested as well, significantly contaminating the sample. In my experience, roughly half of all the detected peptides in the sample were from streptavidin, and on average they were more abundant than the sample peptides, swamping the signal and obscuring low-abundance peptides.

Less trypsin ? Othervise you can try those : http://www.bioclone.us/Monomeric-avidin-magnetic-beads-particle-resin-matrix.html.

Also it depends what protocol of on-bead digestion you use.

I do it like that:

1. Denature in 2m Urea Buffer with DTT

2. Iodacetamide

3.Trypsin (optional DTT to acetamide) 2h

4. fuge, transfer super to new eps

5. denature buffer again, incubate some time, fuge and mix super with the first round super.

6.a bit more trypsin. overnight

7.FA

8. c18 stagetip

For me it worked. YOu still get streptavidin, but it doesnot interfere a lot.

I adopted it from here

http://www.epigenesys.eu/images/stories/protocols/pdf/20130418141114_p61.pdf

Thanks, I'll try this.

I'm curious how much target comes off from the first step (urea, DTT, trypsin) versus the second step.

I haven't found any data about whether urea at room temp will elute target.

There is some evidence that reducing agents at room temp will elute some target:

http://forums.biotechniques.com/viewtopic.php?f=2&t=1249

http://www.ncbi.nlm.nih.gov/pubmed/10023535

Trypsin digestion goes essentially to completion after 2 hours.

http://www.ncbi.nlm.nih.gov/pubmed/16512685

Actually, i am curious about that as well. Have not done that yet. If you will, please share. I want to try to do the same on-bead digestion with rapigest this week, but i will get the data somewhere in august.

Keep in touch!

In my experience, Rapigest incubation at 37°C does not elute very much biotinylated target. See my other conversation thread here: 

https://www.researchgate.net/post/How_can_I_elute_a_target_from_a_biotinylated_antibody_without_breaking_the_avidin-biotin_bond

SDS at room temp doesn't elute trypsin. Maybe Rapigest won't either.

http://www.ncbi.nlm.nih.gov/pubmed/15274123

So, i have done now Rapigest 30min 60°C with DTT in ambic and after i either digested 1h (with 100ng of trypsin with CaCl2) on-bead, or super from beads (fuged down, took a new eps for super). I will have the result in the middle of august. I think the amount of pulldowned protein in my set up is less than 10ug, so i hope it is sufficient amount of trypsin. I want to believe that after reduction and alkylation with surfactant i will get enough eluted protein, but.... results will show...

Got my data. A lot of streptavidin still. (second approach- worked a bit better.)

Now i am going to try, eluting with 95% formamide/10mMEDTA or 8M guanidine ph1,5. The next question about that, whether i can digest my proteins in formamide, or if not, how to get ride of FA. I have no other idea than HPLC cleanup.

It would be perfect if i could use FA with microcon 30kd CW filters (than, you can just go trough FASP protocol) but in their manual they mention that these mebranes are not reccomendet to use with foramide.

Kristian, there is one more point. I have got an answer from Anderson, David Eric (NIH/NIDDK). Hope he wouldnt mind if i post it.

Here are few things to think about;

(1) Make sure that you are "saturating" your beads. Given that your target essentially binds to streptavidin 1:1 (or 1:2 whatever it is) it may be that you simply have strong streptavidin peaks because you have a hundred times more beads than you need. You can establish the right proportion without doing mass spec. (SDS/PAGE etc.). Watch a signal from your bait, use a set amount of beads, and "titrate" in individual samples the amount of bait to the beads. You want to be in slight excess unless you are doing anything where you assume complete capture.

(2) I believe what I did last time people needed to elute in this is we determined that we didn't need the bait in the data set and we eluted with 100mM Tris 8M urea. Pretty much everything but the biotin streptavidin interaction is disrupted by this solution and then you leave the streptavidin behind.

So now i will dive into bead amount "titration"

So i tried Formamide(95%, 10mM EDTA plus free biotin) elution and 8M Guanidine ph1,5 elution. Worked perfect. No streptavidin peaks whatsoever. Problem solved. If you want Kristian, i can upload a chromotogram. Both elute pretty good, but formamide better though.

After elution i just performed FASP, worked well.

Fantastic! I'll try that out.

I keep them 70 C couple of minutes (2min) , before taking the super. Dont forget biotin!

Hi Kirill,

Your answers are very interesting but I was just wondeing how did you perform FASP when formamide is not compatible with filter membranes? Is there a specific brand that you used? I am looking for alternative elution methods that are mass spec compatible and bumped into your duscussion. I hope it is not too late to get an answer.

Thanks

Noo its not too late.

The thing is that i have tried both Formamide elution and Guanidine, and both worked fine. Even more formamide gave better results then guanidine(which is compatible with microcon devices). So sometimes thing that are not recommended still work...

Hope it helps...

The only thing which will help you to get rid of a background is extensive washing, or/and appropriate control.

 What concerns biotin concentration : it doesnt really matters for elution ; i have tried from 10mM to 40mM and have not seen any difference.  In the end i have been just dropping random amount of biotin and it worked fine. I am pretty sure that if you go down to uM concentrations it will still be enough.

Good Luck!

Kirill

According to the attached paper "The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures", the elution of biotinylated molecules from streptavidin beads can be accomplished by briefly heating in deionized water for short periods of time (80 C in ddH2O for

Hi Ian,

I've also seen that paper, but I haven't seen anyone else cite the method. Have you tried it? Do you know if it works for biotinylated proteins?

No, I haven't had a chance to try it out for myself, and I don't know how it would work for proteins. I typically elute by boiling in SDS sample buffer for proteomics experiments, and haven't had any issues re: interference from the eluted (strept)avidin.  

I'd be surprised if you weren't eluting streptavidin by boiling in SDS. What kind of beads are you using? Are you adding streptavidin to your protein database when you search your proteomics data? Rybak et al (linked below) show that boiling streptavidin in SDS breaks apart the streptavidin tetramer, releasing significant amounts of avidin monomer into the sample.

On further investigation, I have found one paper that used heated water to elute biotinylated proteins (linked). Though I wonder how good recovery is, since many proteins (e.g. membrane) won't be soluble in pure water. Also, heating proteins above their melting point in the absence of solubilizing agents is known to cause them to denature, form aggregates, and crash out of solution (which is the basis for thermal shift assays, e.g. PMID:23828940).

http://www.ncbi.nlm.nih.gov/pubmed/15274123

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734586/

Yes boiling in SDS does elute streptavidin monomer, but it doesn't interfere with shotgun proteomic analyses (i.e. by swamping out signal from peptides of interest). You can also build an exclusion list with the common streptavidin peptides if necessary. 

https://www.researchgate.net/post/Can_someone_suggest_a_way_to_elute_biotinylated_proteins_from_avidin_agarose_beads?_tpcectx=qa_overview_asked&_trid=T5GPF70RoeR02bQb54BvpU9B_

Hi Kristian and Kirill, I am also interested in eluting biotinylated proteins and I am also worried about eluting streptavidin. My question is: after eluting with formamide and EDTA what did you do to the samples in order to run them in a gel? Or you just simply took them to mass spect? Another question is, are you using magnetic beads, I'm afraid EDTA treatment would disrupt their structure. 

I tried eluting with 6M Urea + 2M Thiourea + 30mM Biotin + 2% SDS pH 12 in PBS and I couldn't see anything in a western blot, though for sure I eluted everything since if I tried eluting with lamely buffer afterwards I couldn't see anything.

Thanks in advanced!

The easiest way is to do FASP.  Or in your case run them through 30kda (flat one not the V shape) cutoff culumn and just treat with SDS loading buffer straight on the membrane. 

Thanks! I'll read about it and then tell you. Thanks a lot. 

Otherwise, you can just boil the magnetic beads in the sds loading buffer and load to the gel(use magnetic rack to discard beads). Works fine as well.

I normally do it by boiling in loading buffer and it works fine, but I'm afraid I'm not being able to elute the whole sample. Nonetheless, I think it is the most practical way. Thanks for your replies!

Question. Why I need free biotin for the FA, EDTA elution process? And Kirill can you send me the FA, EDTA plus FASP protocol, please

Thanks

I used it simply as a competitor for binding to streptavidin.  I haven`t been doing that for a while already but, protocol should look like that:

1. Beads are resuspended in FA+EDTA+biotin, incubated in +70C for 5 min

2. Beads are fuged down(in case of agarose) or separated on a magnetic rack.

3. Supernatant contains eluted proteins, its loaded onto  30kDa microcon coloumn and followed by FASP.

thanks. Can you forward me the FASP protocol?

And another issue. In point 2 you mention using magnetic beads but aren't they compatible with EDTA?!?

sorry, haven`t tried with magnetic...

Do you have the FASP protocol you used

the best way around this is to use a cleavable cross linker EZ-Link™ Sulfo-NHS-SS-Biotin this way you do not have to take everything off the beads

Hello! Has anyone tried Ian Diner's suggestion on generating an exclusion list containing the peptides form avidin? If so, how could I possibly generate an exclusion list? Thank you!

Hi Kiall, 

Generating an exclusion list is done differently on different instruments, but the essence of it is that you find all of the possible m/z values of peptides from streptavidin (either by calculating them or by finding them from your initial analysis of a streptavidin-contaminated sample), then in the instrument software you instruct the instrument to ignore all of those m/z values. This approach will free up some instrument duty cycle, but it does not address the fact that highly abundant contaminants compete for binding space on the trap column, contribute to ion suppression, and compete for space in the ion trap, all of which will reduce your ability to detect analytes of interest. It's much better to avoid getting the streptavidin in the sample in the first place.

Hi Kristian,

Thank you so much for your response! I was wondering though if you have already successfully employed methods on getting rid of the streptavidin. If so, would possibly share the protocol? Thank you so much!

Since I initially posted this question, I have used two different approaches to avoid streptavidin contamination, depending on my experiment. 1) As recommended by others, for applications where I am labeling proteins with biotin, I use a cleavable biotin tag (e.g. EZlink NHS-SS_biotin) that allows the protein to be released by DTT or TCEP and causes a minimum of streptavidin contamination. (see PMID: 27128092 and PMID: 28759593 ). 2) When the biotin is not cleavable, I use monmeric avidin. Streptavidin is a tetramer, so harsh elution conditions (like boiling) denature the tetramer and release the monomers that are not covalently linked to the bead. Monomeric avidin does not bind biotin as strongly as tetrameric streptavidin, but it is still strong and specific. I have so far only used it to enrich biotinylated peptides after digestion, but it probably works for whole proteins as well. I use Pierce Ultralink avidin (part # 53146) on micro spin columns (Pierce #89879). I load the peptides in 100 mM ammonium acetate (pH~7), rinse 3x with the same, then 3x with ddH2O, and elute with 0.1%TFA followed by 0.1%TFA in 50% ACN. What's nice about this approach is that neither the flow-through (which is in volatile salt) or the eluate require desalting, as both can be dried in a speed vac and re-suspended in your load buffer of choice. I don't know what concentrations of SDS or urea monomeric avidin can withstand if stringent washes are required.

Dear Kristian, I have faced same problem even with cleavable biotin probes. DTT was not enough, so I have boiled the beads with SDS, and have seen avidins in the gel. My major problem is high nonspecific binding in the avidin beads(control). Do you do any pretreatment of the beads to minimize non-specific binding? 

Payal, I have also seen high non-specific binding, even after washing with urea, SDS, and salt. I have not yet found a solution to this issue. To get good cleavage of the S-S linker in the biotin without eluting avidin, try using a lower temperature like 65C, longer reaction time, lower SDS, and/or TCEP instead of DTT.

Have you tried washing with D-Biotin? I've got it in a recent article, hope to try it soon.

To my experience, the protocol that worked better for me has been ON-BEAD digestion with trypsin. The interaction between streptavidin and biotin is so strong that breaking it is nearly impossible, at least in my hands. 

How about crosslinking the streptactin-tetramer before prey binding? Article One-step Purification of Twin-Strep-tagged Proteins and Thei...

The protease-resistant streptavidin might be useful for solving this problem. See the recent article from Jeroen Krijgsveld's team.Article Protease‐resistant streptavidin for interaction proteomics