Question about HILIC-MS

[squall]

Hi All,

Last time, I posted a question about phosphopeptide identifications. At that time, I am using a cell digest, so it's hard to know what peptides are really there, if I don't get good LC-MS/MS data.

Recently I got some "phosphopeptide standard", which I know what is in my sample. First, I tried C18 (LC-MS) and I saw most of the peaks. Then I switch to HILIC column (Amide 80 HILIC), surprisingly I almost lost most of the peak. The problem is not that I got bad peak shape, etc. It's "the peaks are gone".

Actually I got similar problems before which I really can't understand. Currenly I suspect that whether my sample is really dissolved or not. I have 20pmol per phosphopeptide sample, for C18 test I dissolve them in 40ul water with 0.1% formic acid, then I inject 1ul sample for LC-MS analysis. For HILIC, I first add 4ul water(with 0.1% formic acid) to the sample tube, using pipet to mix it. Then I add 36ul ACN (with 0.1% formic acid) inside the tube, sonicate and votex, then also inject 1ul for LC-MS (on-line HILIC MS).

Then I didn't see any peaks. Ideally the HILIC should work better than C18, right? Even the Amide 80 might not be as good as ERLIC for phosphopeptide, how it can lose peaks even from C18?

Is my sample preparation method in HILIC not proper? I really suspect whether the sample got dissolved in this 90% ACN solution. But I saw most papers use 90% or even higher ACN for sample solution, in order to avoid bad peak shape.

Before this sample, I have another single peptide tested on this column using HILIC condition. That works in 90% ACN. But since that sample I have a lot, so I use almost 1ml water to dissolve first, then dilute.
Does this give any information for the above one?

The other question I have is that I always see references adding ammonium salt in HILIC condition. But for the test I did before using simple peptide and small molecules mix, I really can't see any difference adding or not having ammonium salt inside. Is that salt really useful? If yes, why my data shows no difference with regular buffer (water or ACN with 0.1% formic acid)?

Sorry for the long question, I have struggled in this for a long time. Hope I can find some help here.

Thanks a lot

[carls]

The need for buffer vs. a pH modifier (i.e. formic acid) depends on the analyte physical properties. If the small molecules you were analyzing were simple acids then formic acid may be OK. The ammonium is especially useful in moderating cation exchange interactions of basic analytes with the acidic silica surface. Ofcourse your basic analytes must be protonated for cation exchange so very weak bases (pKa <~5) wont necessarily need a competing cation since they likely will not be protonated in the high ACN mobile phase typically used in HILIC.

Since you are now analyzing phosphates its very likely their chromatographic behavior will be improved with a buffer since they show unusual retention behavior with most/all silica based HILIC columns. See my other post on the LC board.