Cysteine Aminoethylation Enables the Site-Specific Glycosylation Analysis of Recombinant Human Erythropoietin using Trypsin.

Recombinant human erythropoietin (rhEPO) is an important biopharmaceutical for which glycosylation is a critical quality attribute. Therefore, robust analytical methods are needed for the in-depth characterization of rhEPO glycosylation. Currently, the protease GluC is widely established for the site-specific glycosylation analysis of rhEPO. However, this enzyme shows disadvantages, such as its specificity and the characteristics of the resulting (glyco)peptides. The use of trypsin, the gold standard protease in proteomics, as the sole protease for rhEPO is compromised, as no natural tryptic cleavage site is located between the glycosylation sites Asn24 and Asn38. Here, cysteine aminoethylation using 2-bromoethylamine was applied as an alternative alkylation strategy to introduce artificial tryptic cleavage sites at Cys29 and Cys33 in rhEPO. The (glyco)peptides resulting from a subsequent digestion using trypsin were analyzed by reverse-phase liquid chromatography-mass spectrometry. The new trypsin-based workflow was easily implemented by adapting the alkylation step in a conventional workflow and was directly compared to an established approach using GluC. The new method shows an improved specificity, a significantly reduced chromatogram complexity, allows for shorter analysis times, and simplifies data evaluation. Furthermore, the method allows for the monitoring of additional attributes, such as oxidation and deamidation at specific sites in parallel to the site-specific glycosylation analysis of rhEPO.

An Affinity-Based Probe for the Human Adenosine A2A Receptor.

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A2A receptor (hA2AR) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA2AR ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA2AR. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA2AR by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA2AR, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.

Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS.

Pregnancy requires partial suppression of the immune system to ensure maternal-foetal tolerance. Protein glycosylation, and especially terminal sialic acid linkages, are of prime importance in regulating the pro- and anti-inflammatory immune responses. However, little is known about pregnancy-associated changes of the serum N-glycome and sialic acid linkages. Using a combination of recently developed methods, i.e. derivatisation that allows the distinction between α2,3- and α2,6-linked sialic acids by high-throughput MALDI-TOF-MS and software-assisted data processing, we analysed the serum N-glycome of a cohort of 29 healthy women at 6 time points during and after pregnancy. A total of 77 N-glycans were followed over time, confirming in part previous findings while also revealing novel associations (e.g. an increase of FA2BG1S1(6), FA2G1S1(6) and A2BG2S2(6) with delivery). From the individual glycans we calculated 42 derived traits. With these, an increase during pregnancy and decrease after delivery was observed for both α2,3- and α2,6-linked sialylation. Additionally, a difference in the recovery speed after delivery was observed for α2,3- and α2,6-linked sialylation of triantennary glycans. In conclusion, our new high-throughput workflow allowed the identification of novel plasma glycosylation changes with pregnancy.