To understand the implications of protein glycosylation for clinical diagnostics and biopharmaceuticals, innovative glycoproteomic technologies are required. Recently, significant advances were made, particularly towards structure-focused N‑glycoproteomic analyses. The mass spectrometric analysis of intact N‑glycopeptides using stepped collision fragmentation and glycan oxonium ion profiling can assist the discrimination between different N‑glycan structures to a large extent. Still, there are weaknesses that current N‑glycoproteomic approaches must overcome. These concern the recognition and handling of incorrect identifications, the identification of rare and modified N‑glycans, and the insufficient glycoproteomic coverage, especially for complex samples.

To address these shortcomings, we have developed an innovative N‑glycoproteomic workflow that provides comprehensive site-specific and structural N‑glycoproteomic data on human blood plasma glycoproteins. The sample preparation workflow features protein depletion, various fractionation strategies, and high-resolution mass spectrometry with stepped collision energy fragmentation. Furthermore, we have established a decision-tree strategy for data validation that significantly improves the description of the N‑glycan micro-heterogeneity.

Our data analysis workflow shows the reliable differentiation of ambiguous N‑glycan structures like antenna- versus core-fucosylation plus modified and rare N‑glycans such as phosphorylated, sulfated, and glucuronidated ones. With this workflow, we were able to advance in the analysis of human blood plasma glycoproteins to concentrations as low as 10 pg/mL. A total of 1,929 N‑glycopeptides and 942 N‑glycosites derived from 805 middle- to low-abundant human glycoproteins were identified. These include prothrombin (110 μg/mL), extracellular matrix protein 1 (0.78 μg/mL), integrin subunit alpha M (1 ng/mL), and cysteine-rich secretory protein LCCL domain-containing 1 (8.2 pg/mL) – protein concentrations reported for human blood plasma.

Overall, the presented in-depth N‑glycoproteomic workflow offers strategies to conduct a refined analysis on complex N‑glycoproteomes, and expands the list of N‑glycans to be assessed as possible blood plasma biomarkers. Applying the strategies shown in this work can significantly enhance our understanding of glycoproteins of clinical interest and contribute to general health care.