Investigation of Erythropoietin Conformation Using HPLC-Chip Based H/DX MS.

Erythropoietin (EPO) is a cytokine hormone that regulates blood cell production. EPO is used for clinical treatment of anemia and renal disease, but may be best known as a performance enhancing drug due to its activity as an erythropoiesis stimulating agent. EPO is a small protein that is heavily glycosylated (40% by mass), and displays large N-linked glycans characterized by high sialic acid content and poly-lactosamine repeats. Thus, it is an ideal model for understanding the effect of glycan modifications on protein dynamics and conformation. We have performed H/DX MS experiments on two recombinant EPOs from CHO cells using a custom microfluidic chip MS system. The analytical platform permits low flow (300 nanoliter per minute) H/DX MS on sample quantities in the 200 femtomole per timepoint range, a sensitivity which is ∼100X higher than that provided by a typical H/DX MS system. Analysis of the EPO samples revealed EX1 type kinetics in two of the four alpha helices of the molecule. These data are consistent with the fact that these two alpha helixes are not involved in either of the two disulfide bridges which stabilize EPO and are required for biological activity. The data thus indicate that these relatively untethered secondary structures take part in rare but extensive unfolding events under native conditions. Comparison of the two recombinant EPO samples revealed similar deuterium uptake profiles at the timepoints tested, likely a reflection of their mutual origin from CHO cells. We obtained high sequence coverage from our H/DX MS experiments despite the high glycan content of the molecule. We conclude that H/DX MS is an ideal platform for comparing extensively post-translationally modified recombinant proteins and that H/DX MS experiments can be optimized for situations where sample quantity is limited.