We are trying to desalt a digest from an IP that has been separated by SDS-PAGE and ingel digested with trypsin (essentially as in Shevchenko et al. 2006). We make stagetips essentially by the protocol by Rappsilber et al. 2007, using C18 material, regular 200 µL pipette tips, and collection in 2 mL Eppendorf Lo-Bind tubes. Buffer A is 1% formic acid and buffer B is 80% acetonitrile and 1% formic acid. We have been using one or two disks made with a 14-gauge needle, which should give a binding capacity of 20-40 µg peptide/disk. We load, wash and elute by centrifugation at 1000-1400 x g (2-3 minutes per spin). Typical volumes of 100-200 µL per spin. Sample is speedvac'ed and reconstituted in 5% FA before desalting.
We always confirm that there is peptide eluted after the digest. The problem is that after binding to the stagetips, virtually all the peptide is in the flowthrough. We have tried several things to troubleshoot, including:
- making sure the filters do not run dry
- conditioning with either buffer B or 100% methanol
- washing gel pieces extensively in 50/50 ACN/water before digestion to remove as much SDS as possible
- speedvac'ing ingel digestion eluates so that no ACN remains before sample is loaded
- running sample twice over filter to load
- one or two disks (note that this is usually much higher binding capacity than the quantity of peptide really demands)
I should note that I have been using this method for a couple of years without major problems. Recently, the quality of my recovery started dipping, and my student is now experiencing the same problems.
I am starting to run out of fixes and would appreciate any tips! I wonder if my C18 material could be degraded (it was bought in 2015), although I consider this unlikely as it should be quite stable.
Probably sounds strange, but it happened to me once:
Check that your speedvac is working properly. I had once a situation in which the vacuum pump was defective and I could smell oil gases upon opening the speedvac chamber.
If something like this is going on in your case, the oil vapors would bind preferentially to the C18 material and block the peptides.
As you say it seems that your peptides are not binding to your C18 disk preparation, so I would check for sample pH and C18 materials. For binding I use peptide sample in acidified aqueous buffer - typically 0.1% formic acid in water. Maybe you should test in parallel a 0.5% acetic acid solution, as described by Rappsilber et al. 2007. I would also test a commercial stagetip, such as Pierce™ C18 Tips from ThermoFisher or Ziptip C18 from Millipore, to control your C18 resin.
Are those peptide that do not bind all from a specific protein or class of proteins? Does the protein/s perhaps have post-translational modifications that interfere with binding?
sometimes I used clean up (or desalt) peptides and using the Stage Tip protocols, obtain a good signal from low gram amounts of starting material.
Hi Rasmus,
What is the length of your C18 material in tip? Apart of disc piece, we always added suspension of C18/ACN to get 1-3 mm total length.
I used “gel loader” tips and 1ml syringe to load/elute peptides (never used centrifugationfor these steps).
You can try commercially available options, if you have money to spend :)
Good luck!
Fedor
If you haven`t yet, I would test the procedure with already clean standard peptides. This way you can determine how much you recover. This you can then indeed compare to commercial solutions. If the recovery is also low with the commercial ones, compare to just speed-vacing the standard. If that`s not the problem, re-make all your solutions and finally re-order the C18.
Hi Rasmus,
I think that the Empora Disks should be stable even if purchased in 2015.
Just to confirm, the pH of the sample should be acidic but not very high amounts of formic acid. After you condition the tip stuffed with the disk(s) with 100% methanol, make sure you rinse them with 0.1% formic acid. The organic solvent (methanol), if still around, will prevent the peptides from binding to Stage tips.
One last thing; make your buffers fresh. Sometimes, if the buffer solution is left uncapped, the acetonitrile or formic acid evaporates and you basically do not have the acidic or solvent capacity of the buffers..
Best,
Hediye.
Hi all, thanks a lot for your replies!
Achim: this is a tryptic digest separated by SDS-PAGE, so I expect to get hundreds of proteins identified. There are no particular PTMs on the peptide we are interested in that should affect this binding, and it certainly shouldn't apply to all the proteins in this mixture. I will check our speedvac! Sometimes it runs a little hot and these evaporated may be a source of the problems!
Fedor: I use regular 200 µl tips (VWR, I think) and they have been ok before. The length of the C18 column is about 2 mm after the tip is packed. I have never tried the syringe method as the centrifuge method have usually worked. I just bought a pack of the Pierce C18 tips and hope they do the trick (for that price they'd better =)
Thanks also for the tips about the ion pairing agent, buffers and standard peptides - I have a lot of things to test now so hopefully one of these fixes will do the trick =)
Have you tried your peptides in a different lab with different equipment?
Amended on 7 May 2019
I must sadly say that Pierce™ C18 Tips from ThermoFisher, Ziptip C18 from Millipore, Sep-Pak® Cartridge (55 – 105 μm particle diameter) from Waters Co., or other similar low-pressure gel or short column or cartridge are not working at all (my unpublished observation using DNA as sample at The University of Tokyo, Yayoi-cho, Bunkyo-ku, Tokyo, Japan, when I have worked as an Arbeiter from 2009 to 2013).
This seems to be due to too large particle size or prticle diameter. Most sample molecules elute without absorption at the void volume.
Therefore, RP-HPLC method (high yield condition, particle size or particle diameter; 5 -10 μm) should be utilized to purify ("please see file; Lysozyme by RP-HPLC" in order to get high-recovery via eluting acidic-phosphate buffer (pH 2.0)-2-propanol-ethylene glycol three-phase gradient system, which firstly has been invented by German Biochemists Wolfgang Mönch and Walter Dehnen).
The above paper used 50 x 4.0 or 4.6 mm I.D. column, but 3.0 x 4.0 mm I.D. column works well to be good loading capacity of c.a. 0.2 mg of protein (my unpublished observation). It is noteworthy that protein samples elute in accordance with alcohol concentrations or hydrophobicity of the sample proteins and peptides in Gradient elution, but not with the column length at all. RP-HPLC is similar to Affinity Chromatography, and RP-HPLC for protein separation should be suitably called as Hydrophobic Affinity-HPLC or C18 Affinity-HPLC or ODS Affinity-HPLC/OA-HPLC.
I have prepared trypsin-treated avidin-affinity column via washing by centrifugation and HPLC washing with solvent B method (please see files; Netherlands Biotin and Lipoic acid Avidin).
Hi Rasmus Ree
I have been following the following protocol
Materials and Reagents:
- Empore C18 material, 100% ACN, Solvent A: 0.1% formic acid, Solvent B: 40% acetonitrile (ACN), 0.1% formic acid
Procedure:
- Pack 10 µL tips with C18 material and pre-wet the column with 30 µL of 100% ACN
- Equilibrate the column with 30 µL of solvent A twice and reconstitute the sample in 30 µL of solvent A
- Load the sample onto the column. Collect the flow through and reload it
- Wash the column with 30 µL of solvent A twice and elute the peptides with 30 µL of solvent B twice
- Dry the elute in a SpeedVaC
Try to centrifuge at 100 xg or 500xg
Hope this works!
Varsha
Hi Rasmus, the diameter of the needle that you use (14-gauge) may cut too large disks for the rather thin end of your tips, leading to the formation of crincles where the buffers and your samples can flow through. Do you check your tips for leakage befor you start the procedure? i.e. by giving a short spin at the methanol step and check for the flowthrough.
Best,
Murat
Hi Murat,
yes, we check the tips by running buffer through them for a few seconds, and discard any that let the buffer through too fast, or ones that are very out of step with the rest of the tips in the experiment.
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