When I run MRM (Using RP-HPLC tamden MS/MS) to quantify targeted protein, there always have two peaks from the selected peptide of my pure protein sample (>99.9% Purity) digested by trypsin (Very close RT, almost overlap (
Dear Sir. Concerning your issue about the RP-HPLC Retention time of peptide. One of the initial steps of proteomic analysis is peptide separation. However, little information from RP-HPLC, employed for peptides separation, is utilized in proteomics. Meanwhile, prediction of the retention time for a given peptide, combined with routine MS/MS data analysis, could help to improve the confidence of peptide identifications. Recently, a number of models has been proposed to characterize quantitatively the structure of a peptide and to predict its gradient RP-HPLC retention at given separation conditions. The chromatographic behavior of peptides has usually been related to their amino acid composition. However, different values of retention coefficients of the same amino acid in different peptides at different neighborhoods were commonly observed. Therefore, specific retention coefficients were derived by regression analysis or by artificial neural networks (ANNs) with the use of a set of peptides retention. In the review, various approaches for peptide elution time prediction in RP-HPLC are presented and critically discussed. The contribution of sequence dependent parameters (e.g., amphipathicity or peptide sequence) and peptide physicochemical descriptors (e.g., hydrophobicity or peptide length) that have been shown to affect the peptide retention time in LC are considered and analyzed. The predictive capability of the retention time prediction models based on quantitative structure-retention relationships (QSRRs) are discussed in details. Advantages and limitations of various retention prediction strategies are identified. It is concluded that proper processing of chromatographic data by statistical learning techniques can result in information of direct use for proteomics, which is otherwise wasted. The set of synthetic peptides and bovine α-lactalbumin fragments (18 peptides) was used in the experiment. Theoretical retention times were calculated using Sequence Specific Retention Calculator (SSRC) program. The experimental retention times were measured via RP-HPLC-ESI-MS method using column working with low TFA content. The dependence between theoretical and experimental tR was expressed via empirical equations. The best correlation between theoretical and experimental retention times of peptides containing at least four amino acid residues has been obtained when third order polynomial (R² = 0.9536). Prediction quality for di- and tripeptides was significantly lower. The method described can be applied for cysteine-containing peptides although our sample preparation procedure did not include modification of this amino acid, taken into attention by SSRC program. I think the following below links may help you in your analysis:
http://www.food.actapol.net/pub/7_2_2011.pdf
https://www.ncbi.nlm.nih.gov/pubmed/19160394
Thanks
Thanks a lot for your answer. Maybe I didn't explain clearly my question. My question is whether the environment or conformation of a peptide split the peptide in UPLC/HPLC(very tiny retention time shift)? Or in other possible condition, peptide bound a water molecule and shift a few seconds in HPLC/UPLC?
Best!
I would recommend to check the analysis in full scan mode. Do you have high res. MS? It seems like coelution of other peptide with the same MRM ions...
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