Chromatography Forum

Hi all,

I'm having some problems with my HPLC protocol...
The idea behind it is to reduce cystine with TCEP to its monomers cystein and derivate it with monobromobimane.

The protocol I started with was terrible and so I changed a lot and it looked good so far, but then I decided to change the internal standard from N-acetylcysteine to cysteamine, what comes out much closer to my cysteine peak. When I tried to seperate the early eluting peaks with adding a gradient at the beginning, I suddenly had a splitted peak for my cysteine. On the first runs it wasnt really a split peak. It looked more than two different peaks.
By adjusting the gradient I almost managed to flush both of them out simultaneously, but I'm writing this here now under sample preparation because its not a HPLC problem (even if I try to solve it by adjusting the gradient) but a problem with the sample preparation. Because only this peak is splitted, there is a strong evidence to suggest that during my sample preparation really two slightly different products are generated.

Thats what it looked liked in the beginning:

with peak 1: rubbish peak 2: internal standard peak 3,4: cysteine, rest:rubbish

after adjustments:

(i know I let the column not enough time to re-equibrilate but I wanted to do it fast)


And thats my actual sample preparation:

50µl sample in PBS buffer
10µl cysteamine 0.05mM
6µl TCEP 5mM adjusted to pH 7
30 min incubation 37C
20µl Monobromobimane 3mM in acetonitril
20 min incubation at RT in the dark
20µ acetic acid 10%


Any Ideas what could happen in my assay?

Thank you so much!

Friede

What other corners are you cutting, besides working with a column which is not equilibrated?

Thanks for your great help Mr Müller.
Its a sample preparation question and as long as my preparation doesnt work, I do not care about small changes in retention time. I dont quantify any samples with standard curves like that...

The protocol I started with was terrible and so I changed a lot and it looked good so far, but then I decided to change the internal standard from N-acetylcysteine to cysteamine, what comes out much closer to my cysteine peak.

So you don't see the peak splitting if you use the N-acetylcysteine as the IS? Is there a chance that you have misidentified one of the peaks?

It would help to see four chromatograms (run under the same conditions):
N-acetylcysteine only
cysteamine only
cysteine only
all three together.

that was my first thought too, but in fact (and I forgot to mention that) gives the integration of both of the peaks a standard curve with R > 0.999. So I´m quit sure that both peaks belong to cysteine. I could see the two peaks after I changed the internal standard, but I also changed the gradient... I could imagine that the cysteamine reacts in some way with my cysteine. It is known that cysteamine helps dissolving cystine by building cysteamine-cysteine disulfid bonds, but they should be reduced by the TCEP and without a free thiole group no derivatisation should be possible.
Anyway, my next run will contain NaC instead of cysteamine again and the both of them.

I would kill to have a MS on hand...
Thanks

So more shortcuts?: You changed two things at the same time, IS and gradient?
You don´t have any idea how high the yield of your derivatization is? (You are not sure whether the -SH reacted with bimane or with IS??).
You say that you don´t care about rt, but then ask us to help about peak splitting, or whatever, that involves a few seconds?
Incidentally, I am trying to help people figure out problems rationally, not do the work for them. You may have a solution to your problem if you think about what I said.
This used to be a discussion forum, not a "hey, give me a method" session.

1.) I never asked for a method! Since Im sure that I have two products out of cysteine and bromobimane, I only wanted to know if someone has any idea what could happen in this assay... And I would really appreciate a discussion, but It doesnt help a little when you question everything else that has no relation to my problem.
The two chromatograms I embeded are only to show that there are two products. I made them for nothing else. Its a just a matter of the chemical reactions not about peak splitting. Otherwise I would have asked in the chromatography board.

2.) I changed the gradient because I changed the IS. The new IS peak comes close to the cysteine peak, so I added a gradient at the beginning.

3.) Initially, I start with cystine, which is the disulfid-bond dimer of cysteine. To reduce the disulfid bond, TCEP in surplus is added. TCEP as a reductive agent is strong enough to reduce any SS-bond that could be build between different thiols.
The thiole group is also where cysteine (and IS) get derivatized by the bromobimane to build the fluorophor. Even if the cysteamine reacts temporary with some cysteine, after adding the TCEP all SS-bonds should be reduced and after the reaction with bromobimane the thiole group is save from further oxidation.

Thats 4.) why I'm sure that cysteine-bromobimane bonds are build properly, but that perhaps due to the pH range I'm working in the cysteine-bromobimane molecule can slightly change, and thats what I'm asking for. Any Ideas what could happen?

Thanks

I disagree emphatically on the idea that working off equilibrium, and other shortcuts, not being relevant to your problem. Also, again you don´t know what you are doing as derived from the statement "S-S bond should be reduced". I just tried to find out what gives. I helped a doctoral student derivatize glutathione and do HPLC. I don´t recall any intransigent problems. Too bad though, I don´t remeber the derivatization reagent, it was a complicated derivatization, I later saw simpler ones. I had/have the feeling that problems have long since been worked out.