"jagged" peptide peaks

[hooky]

Hi all,

I am developing an LC method for a peptide with 20 AAs. I've been told there are 5 arg/lys/his

My problem is that the peak is looks jagged and I would say that it must have several coeluting peaks. I have tried different types of chromatography and columns to try to resolve this:

C4/C12/C18 columns with ACN/ 0.1% TFA in various combinations/gradients
SCX pH 2.5 early eluting, poorly retained
IP with hexane sulfonate on C18,

I have tried diluting the samples in water, 6M urea, 0.1% TFA with similar results.

all of the chromatograms suggest several coeluting peaks (4 or more).


Is it possible that this is an artifact? (eg. non-homogeneous charge states? or some kind of isomerism?)

If it is an artifact, what else could I try to resolve this? The chromatographies seem reproducible, so I'm thinking that perhaps the sample prep may be the real issue...

Suggestions?

cheers

[Andy Alpert]

Hooky:
It isn't clear why the peptides eluted early in SCX. Normally a peptide with this many basic residues would require about 0.5 M salt for elution. What SCX column and mobile phases were you using? Anyway, try ERLIC. ERLIC has the best selectivity I've seen for peptides with numerous basic residues. Here's a link to the relevant paper: http://pubs.acs.org/doi/pdf/10.1021/ac070997p
Initially, I'd recommend running isocratically with 20 mM sodium methylphosphonate, pH 2.0, with 65% acetonitrile. To get sodium methylphosphonate, dissolve some methylphosphonic acid in water and add NaOH until you get to pH 2.0.

[Kostas Petritis]

Assuming you have the resources, the first obvious thing to do is LC-MS to see if your peptides have the same mass and fragmentation... What wavelength are you detecting those?

I agree with Andy that the peptide should had been very retained in SCX if it contained so many basic residues...

[samihat]

I agree with Andy too