Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity

Yang Yang, Fan Liu, Vojtech Franc, Liem Andhyk Halim, Huub Schellekens, Albert J R Heck

Research output: Contribution to journalArticleAcademicpeer-review


Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resolution native mass spectrometry and middle-down proteomics, to analyse this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addition unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quantitatively assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.

Original languageEnglish
Pages (from-to)13397
JournalNature Communications [E]
Publication statusPublished - 8 Nov 2016


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