Stable peptide quantification is essential for assay development based on LC-MS, especially in low protein concentration ranges.
Do you recommend quantification of all fragment ions obtained from one precursor or would you rather quantify the most intense fragment ion and use 2 additional fragment ions for correct peptide identification?
Dear Viktoria Anselm,
Your question is difficult one. And the answer is dependent upon your experimental design. Since ionization in LC/MS is not a stable factor, depending on the matrix of the sample, to name but one parameter, things will essentially boil down to having a reliable and stable internal standard. I would recommend to add a 13C stable isotope of your potein of interest to your sample. This may not be available, but then just grow your cells on 13C enriched glucose. Kind regards, Harrie Verhoeven
Dear Harrie A Verhoeven,
thank you for your answer! Fortunately, I am using heavy labeled peptides for absolute quantification of my targeted peptides (in blood plasma samples). Still, I am not sure which approach is better/recommended:
1) focus on quantification of one specific fragment ion for the precursor and use additional 2 specific fragment ions for correct identification of this precursor (in total 3 specific fragment ions)
2) use all fragment ions of a precursor for quantification
3) use all fragment ions for quantification which have higher m/z than their precursor (since fragment ions often have one charge less than their precursor, there are often at least 3 fragment ions fulfilling this requirement)
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