Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering.

David P Cowcher,Royston Goodacre,Lorna Ashton,Victoria L Brewster,Volker Deckert,Tanja Deckert-Gaudig

doi:10.1021/acs.analchem.5b03535

Abstract

The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.

Highlights

I t is estimated that glycoproteins account for almost twothirds of all protein species,[1] and with the level of research and investment into protein-based therapeutic products ever increasing,[2] the accurate characterization of post-translational modifications (PTMs) is vital for therapy
At first the tip-enhanced Raman scattering (TERS) tip was scanned through the laser spot and positioned at the location giving the highest in Figure 1 shows the structure of the protein and its associated reflection in the optical response image
We have demonstrated for the first time that TERS can be used to distinguish between glycosylated and native forms of proteins

Summary

■ RESULTS AND DISCUSSION

The RNase model proteins chosen for this work were both the native form, RNase A, and the glycosylated form, RNase B, and are available commercially in high purity. Any glycan specific information.[14] With the exception of the 300−780 cm−1 region that includes any artifacts from the removal of the Si fundamental peak, the AUCs show little difference between the two forms of the protein (the two medians are very close as are the IQRs), indicating that we may be able to classify the proteins based on features within the “sugar regions” of the spectra. These six sugar regions (Figure 3, Table S1) were isolated and PCA performed on each region individually. With this level of control in place, it would become more likely that structural changes as a result of incorrect glycosylation would be detectable

■ CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES