What influences peak intensity in peptide mapping?

[Woody1976]

I am currently developing a peptide mapping method by RP-UPLC and have problems with two late eluting peaks containing two large peptides (around 10 kDa) that are not "robust" in terms of peak intensity but with stable retention times whereas the other peaks in the chromatogram are stable in terms of both retention times and peak intensities. I have tested different sample prep but all have issues with the two last peaks in the chromatogram and I suspect more and more that the problem layes in the chromatographic parameters. I am using a Waters H-class Uplc with a Kinetex C18 1.7 um column with 0.06% TFA in the A solvent and 90% acetonitrile/0.055% TFA in the B-solvent. My question therefore is: what influences the peak intensity and why only some of the peaks?

[Kristof]

If they are partial digestion products (>1 miscleavages) they could be quite sensitive to variation in sample preparation conditions, and sample storage conditions and time.