- A brief introduction to the feasibility of estimating the retention time of unmodified peptides
- Reproducibility of peptide retention time.
- Retention time estimation method
- Indeed, subsequent studies have shown that even for short peptides, different retention coefficients are obtained when amino acids are located at different positions in the sequence.
A brief introduction to the feasibility of estimating the retention time of unmodified peptides
The results can be repeated by HPLC. Except for a small change between runs, the same peptide elutes at about the same time when the sample is reanalyzed under the same experimental conditions.
The consistency of elution time indicates that the peptide is involved in a definite interaction in the separation process, which only depends on the experimental setup used and the characteristics of the peptide itself (antoles bioassay shows that reproducibility only occurs in similar experiments and does not extend to other experimental conditions and instruments).
The properties of peptides depend on their amino acid sequences. Therefore, given the chromatographic settings, the estimation of the retention time of peptides should provide the required information for the retention time of the sequences.
Reproducibility of peptide retention time.
But in fact, this prediction model is not as direct as it seems. For example, an important part of this model is the three-dimensional conformation of peptides.
Although the current structure prediction methods are accurate in predicting the three-dimensional conformation of short amino acid chains, this method will not be able to flexibly model various conditions of chromatographic experiments.
Many peptides may change their determinacy according to the amount of organic solvent in the mobile phase, which may play an important role in the retention time of each peptide.
Therefore, most chromatographic processes are considered to be black boxes.
In other words, based on these observations, various methods for estimating retention time are proposed. One of them is an index based approach. The main idea behind this approach is shown in the figure.
Retention time estimation method
Index based method for retention time estimation
The index based prediction method aims to evaluate the effect of each amino acid in the sequence on the peptide retention time. Each contribution of amino acids is usually called retention coefficient. A group of retention coefficients forms a retention index.
Once the retention index of a given chromatographic system is obtained, the retention time of peptides can be estimated by simply summarizing the retention coefficients of the constituent amino acids.
One way to realize this idea is to use a set of 25 short peptides and their observed retention times to derive each amino acid and terminal retention factor present in the sequence.
The coefficients are calculated in the form of Iterative Regression, and only the amino acid composition of the peptide is used to estimate, without any information about each amino acid position in the sequence.
However, since previously reconstituted versions of the same peptide sequence have been separated on the RPLC column, Antonius biology suggests that other parameters are needed to estimate the retention time of longer peptides.
Indeed, subsequent studies have shown that even for short peptides, different retention coefficients are obtained when amino acids are located at different positions in the sequence.
In that case, a different retention factor is required for each position in the putative peptide sequence. Although the exact chemical mechanism of this observation has not been fully understood, many studies have identified individual factors that alter the amino acid retention coefficient.
For example, it has been shown that the retention contribution of amino acids is affected by the overall hydrophobicity of the peptide, the number of positive charges or the presence of secondary structural elements.
In addition, changes in chromatographic settings, such as changes in the composition of the mobile or stationary phase, also alter the elution pattern of peptides.
Some studies have shown that the interaction involving helix structure is highly complex, and more complex models are needed to consider the effect of this interaction on peptide retention time.
The limitation of index based methods is that they are optimized for the estimated retention time of a specific chromatographic setting.
Therefore, for the experimental conditions which are different from the optimization algorithm, their prediction ability will be reduced.
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