(M1130-03-21) Mitigation of Non-Specific Adsorption of Peptides using Inert Surfaces

Purpose: Non-specific adsorption can be a major challenge in analysis of biomolecules when using traditional stainless steel high performance liquid chromatography (HPLC) systems. Many biomolecules have electron-rich functional groups that interact with electron deficient metal oxide layers on stainless-steel tubing throughout the system flow path. One example of this is the phosphate group on phosphopeptides. This can result in broadening peak shape, decreased recovery, and poor repeatability. There are some common mitigation techniques such as passivation through repeated injections prior to sample analysis or use of mobile phase additives, but these can be time consuming, require repeated use to maintain performance, and are not compatible with mass spectrometry (MS). As an alternative, technologies have been developed that are designed to mitigate the effects of non-specific adsorption. Different materials such as MP35N and PEEK are used throughout the flow path, and systems, columns, and vials can use other inert surfaces to prevent any sample to system interaction. In this study, two modern HPLC solutions with bioinert qualities are compared with a legacy stainless steel HPLC system by examining the performance of a phosphopeptide standard. System performance was characterized using retention time standard deviation, USP tailing, and area % relative standard deviation. Bioinert systems providing increased performance are expected to see improved repeatability and recovery, and sharper peaks in the form of decreased tailing.<

Methods: Three total systems including two bioinert HPLCs and one stainless steel HPLC were used. Each system used 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile as mobile phases A and B respectively, and used a gradient from 5% to 25% B in 18 minutes. A column designed for peptide analysis was used (4.6 mm x 100 mm, 130Å, 2.5 µm). Column temperature was held at 60 °C and sample temperature was held at 5 °C. UV detection was performed at 5 Hz at 214 nm. A phosphopeptide standard containing four peptides was used as the representative sample. Vials containing 1 nmol of each peptide were reconstituted using 100 µL of mobile phase A and were pooled to yield sufficient volume for all systems then distributed to minimize variability. Injections were made at 25 µL.

Results: Phosphopeptide standard injections were run on the three systems. As shown in Figure 1, peaks on the bioinert systems show a larger response and a sharper peak shape. The overlay also shows a higher level of repeatability on the bio system. This is confirmed quantitatively in Figure 2 where bioinert system X has a maximum retention time standard deviation of 0.003 minutes and bioinert system Y has a maximum standard deviation of 0.010 minutes, while the legacy system had a minimum standard deviation of 0.014 minutes. The fourth peak in the chromatogram, t43pp, is a doubly phosphorylated peptide while the other three are singly phosphorylated. The doubly phosphorylated peptide is more prone to non-specific adsorption due to the presence of two phosphate groups and thus would be expected to be more affected by to sample to metal system interaction. This can again be observed visually in Figure 1 where the peak height of the t43pp peak has the largest difference when compared to the result on the bioinert HPLC system. The quantitative results of the t43pp peak are summarized in Table 1. The two bio systems have similar height and area results, and both have an area % RSD under 2.5%. The legacy stainless steel system has raw peak height and area results of about one third of the two bioinert systems, and an area % RSD of 7.85%. USP tailing was also most impacted on the doubly phosphorylated peak, where the legacy system had a result of 1.60 while the two bioinert systems had values of 1.25 and 1.37.

Conclusion: The efforts to mitigate non-specific adsorption using full system solutions were found to be successful. The chromatography shows sharper peaks and better repeatability on the bioinert systems than the stainless-steel system. Retention times were more reproducible on both bioinert systems for all peaks, and the quantitative differences were most noticeable for the doubly phosphorylated peak. For that peak, the raw height and area counts were significantly lower on the stainless-steel system, while the area % RSD and USP tailing were higher than the bioinert systems. Overall, the modern bioinert systems were more capable of producing strong results for the phosphopeptide sample. Continued improvements of instrumentation plays a key role in advancement of biomolecule analysis, and the mitigation of non-specific adsorption demonstrated by these systems shows just one example of how this is possible.


Figure 1. Overlay (n=6) of injections on Bio HPLC System X (top) and Legacy HPLC System (bottom). Retention time differences are due to different dwell volumes between systems.


Figure 2. Retention time standard deviation results across for all peaks across the three systems used throughout the study.


Table 1. Height and area results (n=6 injections) for the doubly phosphorylated t43pp peak across the three systems used throughout the study.