Cysteine Aminoethylation Enables the Site-Specific Glycosylation Analysis of Recombinant Human Erythropoietin using Trypsin.
Steffen LippoldAlexander BuettnerMatthew S F ChooMichaela HookCoen J de JongTerry Nguyen-KhuongMarkus HabergerDietmar ReuschDana L E VergoossenNoortje de HaanPublished in: Analytical chemistry (2020)
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.
Keyphrases
- recombinant human
- mass spectrometry
- liquid chromatography
- electronic health record
- high resolution mass spectrometry
- tandem mass spectrometry
- high resolution
- hydrogen peroxide
- transcription factor
- gas chromatography
- optical coherence tomography
- dna binding
- high performance liquid chromatography
- capillary electrophoresis
- fluorescent probe
- simultaneous determination
- deep learning
- data analysis
- solid phase extraction