By Anonymous on Sunday, June 20, 2004 - 04:22 am:

Hi,

I am analyzing small glycopeptides in serum and wish to perform some enrichment prior to purification. I understand that Oasis is supposed to retain most compounds but i have had no success with this product.

I have tried the generic protocol (load sample -aqueous then wash with 50% methanol and elute with 100% methanol) with oasis. The compound eluted in the initial load and wash step.

I then tried mixing serum with MeCN and loaded the supernatant onto the oasis cartridge but i was unable to detect the glycopeptide in the eluent (water). It turned out that the glycopeptide was in the insoluble pellet after addition of MeCN.

Can someone suggest what is wrong and how i rectify the problem

Are glycopeptides poorly soluble in organic solvents and will this affect the seperation of this compound when performing a HILIC mode of chromatography?

Does anyone have experience with solid phase extraction for this type of compound?

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By Uwe Neue on Sunday, June 20, 2004 - 12:04 pm:

You are better off using the Oasis MCX cation exchanger and a standard protocol. Acidify your sample to obtain a positive charge of your analytes (pH about 1). Load the sample onto the ion-exchange cartridge. You can now wash it with methanol, and then elute at neutral or basic pH with a methanol concetration of your choice. You may also do the exact opposite protocol using the Oasis MAX anion exchanger.
We have done separations of polar peptides and of carbohydrates in HILIC mode, but I do not have an example of glycopeptides. I do not think that there is a fundamental problem with this, though.

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By Anonymous on Monday, June 21, 2004 - 08:21 pm:

Dear Uwe,

With oasis catridge, there would be no retention of my compound?

I have not tried the OASIS MCX but i have tried both cation and anion exchanger (Q and SP using aqueous buffers) but there does not seem to be any retention of my compound? Will MCX work?

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By Anonymous on Monday, June 21, 2004 - 09:06 pm:

Glycopeptides are very polar, and you may not get good retention by reversed-phase.

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By maris on Tuesday, June 22, 2004 - 07:18 am:

Decide the C30 SPE - unexpected good retaining of polar compounds was observed.

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By maris on Tuesday, June 22, 2004 - 07:21 am:

sorry, don't decide, just consider

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By Uwe Neue on Tuesday, June 22, 2004 - 11:18 am:

Peptides can in general be retained on the Oasis packing. Your compounds are glycopeptides, which makes them much more polar, and also the peptides are small. This may be the primary reason why you do not get retention on the Oasis packing. This is the reason why I have suggested an alternative technique.
The solubility of most compounds in HILIC mode is low, but with sensitive detection techniques, this is usually not a problem.

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By Anonymous on Wednesday, June 23, 2004 - 02:08 am:

Thank you for the advice.

Actually i am looking for a method to scale up the purification of this comppound but so far had little success.