I'm carrying out the identification of some natural peptides by mass spectrometry. The extracted samples are diluted and rich in salts. Nowadays I'm desalting the samples by HPLC-RP18, but it could take ages if you start from relative big volumes, as I do.
Some previous works in the field suggest the use of SPE for peptide desalting, but without going into details about suppliers and cartridges details. I had used strataX from phenomenex, following vendor's instructions, but I lost most of peptides in the non-bounded fraction or in the washing steps.
Any suggestion?
Reverse osmosis membrane use will solve the issue.... Millipore people can give exact membrane details on contact
First of all, is there any specific reason you didn't use short column like Sephadex or Bio-Gel or similar on HPLC?
If you continue with solid-phase, you can try C18 SPE cartridges.
Ultrafiltration can be used as an alternative technique.
Go with C-18 SPE cartridges. You can purchase it from Waters. This will give you better results.
Gud luck!!!
:)
this is what U need :)
if not helps, i have another protocol.
Please refer to the paper
fast and efficient purification
of synthetic peptides by solid-phase extraction by w. Kamysz it will help you
it almost this same mathods (exept sorbent - C8)... thw question is how big (Da) peptides collegue Amaury Pupo have - this same what Mr. Kamysz et all. synth.?
Better than C18 Sep Pak type of stationary phase is to use Oasis HLB cartridges - they are polymeric and don't suffer from running dry. You will need to ensure you load your peptides uncharged though or you may suffer losses.
I would suggest to use OASIS ion exchange cartridges & also try 300°A C 18 column for proper resolution.
Try with Supelclean™ LC-4 (Wide Pore) SPE,should be working fine.
Thank you all for your good advices.
@Rohit Soni and @Kiran Taimoor: Yes, I will try C18 SPE cartridges, maybe the strataX system was a bad choice for peptides (although announced as superior to C18).
@Leo Phillips, @Jeff Horsman, @Shailendra Gupta and @Karl-Erik Gustavsson: I will try your suggestions, thank you very much.
@Mariusz Dziadas: I am working with high sample volumes, > 10 mL, so small tips won't work here. Thanks for your protocol anyway.
@Rohit Soni: Nice paper. Although I prefer to use some vendor cartridge rather to build one myself.
@Krzysztof Bielikowicz: The mass of my peptides range from 0.9 to 2.5 kDa.
Sorry. I had not seen all the replies.
@Krzysztof Bielikowicz: Thank you. I will try C18 SPE cartridge from Phenomenex. I will try this first.
@Esmaeil Sadroddiny: I work in the mL range, and I don't know any zip-tip with so high charging volume.
@ Dr.B.k Chakravarthy: Some of my peptides of interest are really small, with 8-9 residues, so I guess I will lost them with a reverse osmosis membrane.
Thanks a lot.
Hi Amaury,
I was just wandering which method suited you best for desalting the peptides.
I am facing the same problem...
Hi Swati,
For relatively small volumes I keep using HPLC-C18, which give me by far the best recovery and purity. The "small" part depends of you column capacity and the maximum flow you can achieve with your HPLC system. For larger volumes I used C18 SPE cartridges with success. I recommend you to test you are not loosing your peptides with whichever cartridges you choose. If you have access to a MALDI or ESI MS then you should check if you are loosing peptides in the non-bounded fraction or in the washing steps.
Good luck!
Hi Amaury,
I'm facing more or less the same problem and I want to use the cartridges...In our case I have Agilent Bond Elut C18 1 mg/3 mL cartridges. Could you please send me the protocol that you used for peptides...I found many on proteins and I don't want to loose the small amounts that I gathered so far.
Thanks a million for your help in advanced
Just to give you an alternative (and in case you have very small polar peptides) you might want to consider graphitized carbon SPE.
Hi Amaury Pupo ,
I am using C-18 spin column for peptide desalting following vendor's protocol, but I failed to bind peptide onto C-18 column. I removed 5% ACN in the sample buffer and it didn't help. could you give me some suggestions? Have you tried C-18 spin column before? Is C18-cartridge better than spin column?
Thanks