Re: MS: determination of citrulline containing peptides
Katheryn Resing (Katheryn.Resing@Colorado.EDU)
Mon, 16 Aug 1999 10:48:02 +0000
If you get a helpful answer on this one, I'd love to hear about it. I
tried this, and I decided it was impossible with our instruments, and our
sample.
It sounds like you will have random citrullination, so you will be
looking for a bunch of low yield products (I had the same situation).
Furthermore, your citruline sites will not be cleaved by trypsin, so you
will be looking at larger peptides (equivalent of the incomplete cleavage
products) or you will need to shift to another digestion method (I tried a
couple, but the amino acid composition of my protein wasn't very good for
alternatives). Unfortunately, the peptides will only be one dalton larger
than the original peptide, so they will be hard to identify unless your
instrument has sufficient resolution (don't forget there may also be the
normal incomplete proteolytic products).
On the ESI, if I'd had a lot more sample, so that I could do MS/MS
at unit resolution, I probably would have been able to make a case. If
there had been no incomplete cleavage product at the site, it would have
helped, because as it stood, I had to make a rather complex argument.
On ESI, don't forget the charge will change, so the peptides may
just disappear from view, ours was fairly large, but the doubly charged
were around 1300 and 1400 Da.
On maldi, our putative citrullinated peptides wouldn't fly in the
complex mixture or in the purified sample (HPLC fractions which I thought
had the peptide in it, from the ESI data). They were low intensity ions,
when I lyophilized to concentrate, I lost a lot of the sample, so there
wasn't a much better maldi signal. In theory, the maldi could have given
me the resolution to tell the incomplete product from the citrullinated
product, but the peptides were too large for PSD anyway, so I didn't pursue
the maldi route.
I tried MS/MS, but it was low intensity, and didn't get very good
fragmentation. MS/MS of a citrulline containing peptide might produce a
different set of immonium ions, than the arginine containing incomplete
proteolytic product will. But you'd need some studies with standards to
show that, I gave up before getting that far.
Katheryn Resing