HPLC method for hydrophobic peptide from antibody digest

[JJG]

Does anyone have any suggestions for separating and/or recovering very hydrophobic peptides by HPLC on a method which is MS friendly? We have been trying to find a 54 amino acid peptide from an IgG1 Reduced LysC digest, and have not been able to find it on a reversed-phase column C4 thru C18, even at temperatures up to 80C.

We've been running TFA gradients in ACN and IPA, up to 95% and don't see it. It could be sticking to the column, or maybe the vial, or falling out of solution. Or a combination of these variables.

We have been able to see a small peak, which co-elutes with other peaks, on a PLRP-S column. It elutes in about 38% ACN, shortly after all other major peptides. It is only a fraction of the peak height we expect to see! This suggests poor recovery from the vial. Any suggestions for better recovery of hydrophobic peptides?

I really only care about recovering/analyzing this one peptide, but if I can develop a method which contains the other peptides, that's okay too. Any suggestions for other separation conditions which I might be able use to resolve/recover the peptide from the column? Ideally, they would be MS friendly.

Thanks!

[pph31]

MeOH and formic acid or ammonium acetate

[danko]

Try alkali, or neutral conditions - ammonium carbonate buffer for instance.

Best Regards

[Andy Alpert]

Why not try HILIC? It will simply ignore the hydrophobic residues and promote retention through whatever hydrophilic residues the peptide has. You can use a blend of propanol and ACN as a better solubilizing solvent for hydrophobic peptides. If necessary, include 50 mM hexafluoro-2-propanol in the sample solvent too. Some years ago we presented an analysis of beta-amyloid peptide(1-43) this way, using a PolyHYDROXYETHYL A column. That peptide has solubility comparable to that of nylon. Reversed-phase only works at very high temperature. HILIC worked fine at room temperature.

[magicdice]

Hi Andy
I am trying to purify the beta-amyloid peptide (1-42) and saw your post and I want ask you about you analysis of beta-amyloid peptide (1-43) using to PolyHYDROXYETHYL To column and/or reverse phase (C18 column). I have not been able to solubilize this peptide and it interests to me to know that strategy used.

Greetings and thanks

[Bryan Evans]

Presto FF-C18 (2um non-porous ODS) would be a great column for this application.

[Andy Alpert]

Magicdice:
This was presented at the Protein Society Symp. in 1994, and was one of our first applications of HILIC. Beta-amyloid sequences longer than (1-41) are about as soluble as nylon. Accordingly, synthetic (1-43) was dissolved in the minimum amount of pure hexafluoro-2-propanol (HFIP). The column was PolyHYDROXYETHYL A (200x9.4-mm; 5-um, 200-A) with a flow rate of 0.8 ml/min and detection at 215 nm. The mobile phase was 50% acetonitrile (ACN), 0.1% TFA, and 0.4% HFIP (omission of the HFIP led to severe problems with solubility). The amyloid peptide solution was slowly diluted with mobile phase and injected. The amount of ACN was too low for significant hydrophilic interaction (you'd need about 60% or more for that), so the conditions reflect a combination of SEC and a minimal amount of hydrophilic interaction. Aggregates of the amyloid peptide eluted at 6.1'. The amyloid (1-43) eluted at 7.1', well-resolved from the neighboring peaks. A truncated sequence eluted at 8.5' and p-cresol eluted at 10.2'.
If I were to do this work nowaways, I'd use a blend with 45% propanol, 30% ACN, and the same amount of TFA and HFIP [note: the effects of chaotropes like TFA and HFIP are additive]. This would afford good retention so you could use a more conventional combination like a 200x4.6-mm column and a flow rate ~ 1 ml/min.

[mbreslav]

I have both analyzed and purified synthetic hydrophobic peptides and peptidomimetics up to 12K DA using Zorbax SB C18 300A material at 60C. Also there are papers from Amgen on analytical C18 HPLC of denatured antibodies at high temperatures with N-propyl alcohol in a mobile phase. I dissolved hydrophobic peptides in DMSO and used a variety of at-column dilution techniques.