Human Blood N-Glycoproteomics

Human Blood N-Glycoproteomics


Glycosylation is the main post-translational modification in human blood plasma proteins. Alterations in the N‑glycosylation of human blood plasma proteins can reflect physiological and pathophysiological changes. Furthermore specific N‑glycosylation changes can occur only at particular N‑glycosylation sites. The core technology for detecting these site-specific changes is glycoproteomics. Glycoproteomic analyses can be powerful yet a high performance is hard to achieve due their complexity. Therefore, our group focuses establishing sample preparation and data workflows that maximize the site-specific structural elucidation of N‑glycans found on complex samples such as blood plasma.

Aim of the project

To expand the understanding of micro- and macro-heterogeneity of N‑glycosylation of proteins of clinical or biopharmaceutical interest. To this end, new methods must be developed and further adapted to overcome the complexity of each sample. Therefore, a sample preparation and data analysis workflow were developed for in-depth N‑glycoproteomic analysis.

Sample preparation and data analysis workflow for in-depth N‑glycoproteomic analysis

Due to the complexity of human blood plasma proteome, the in-depth N‑glycoproteomic analysis of this sample encounters two main hurdles. The first is that a few high-abundant proteins cancel the signal of hundreds of proteins distributed at lower abundance. The second is the heterogeneous and unpredictable nature of protein N‑glycosylation. That is to say, if one protein has three N‑glycosylation sites and ten different N‑glycan structures attached to each site, then 1,000 different glycoforms only of this protein will be observed. Thus, massive amounts of information must be acquired and analyzed to elucidate the micro- and macro-heterogeneity of multiple glycoproteins. On the one hand, we established a sample preparation workflow that generates several protein fractions and integrates a broader data acquisition strategy (Figure 1). On the other hand, a sophisticated glycoproteomic data analysis workflow, which includes an N‑glycopeptide validation strategy, was also developed (Figure 2). By coupling these workflows, we expanded the detection of the low-abundant N‑glycoproteome.

The low-abundant N‑glycoproteome detected by us

Several results were obtained employing the established N‑glycoproteomic workflows to a blood plasma standard sample (Figure 3). In the first place, a higher number of glycoproteins within the middle- to low-abundant blood plasma concentration range were identified. In the second place, rare N‑glycan compositions containing glucuronic acid, sulfation, phosphorylation, and even rare N‑glycan building blocks, were detected by us for the first time. The rare N‑glycans were detected mainly on glycoproteins within the middle-abundant concentration range, but they were also identified in glycoproteins from the lower abundance range. In the third place, the data acquisition strategy employed and developed in precedent works in our group allowed the structural elucidation of N‑glycans to some extent. As a result, we discovered an N‑glycan structure bearing a rare glycosidic linkage between a sialic acid and an N‑acetylhexosamine. Finally, the data analysis workflow exposed opportunities for improving the data validation strategies and the success of glycoproteomic searches.

Another project that benefited from these workflows was the site-specific identification of a sulfated N‑glycan characterized in immunoglobulin A (IgA) only via glycomic analysis. Our study, integrated an oxonium ion-guided strategy to screen for sulfated N‑glycopeptides from human serum IgA, which typically comprises two subclasses (IgA1 and IgA2). We discovered diverse compositions of hybrid- and complex-type HexNAc-sulfated N‑glycans attached to the N‑glycosylation sites in the tailpiece and in the CH2 domain (Figure 4). Surprisingly, also complex-type N‑glycan compositions with O-acetylated sialic acid were identified primarily in the tailpiece (Figure 4). Human serum IgA is a glycoprotein of therapeutic interest and each of its N‑glycosylation sites affect the effector functions. For example, sialylated N‑glycans on the C‑terminal tailpiece interfere with sialic-acid-binding viruses. We expect that a wider micro-heterogeneity description of clinically relevant glycoproteins, such as IgA, can expand the screening for biomarkers or a treatment options.


Zuniga-Banuelos, F. J.; Hoffmann, M.; Budimir, I.; Reichl, U.; Rapp, E.: New avenues for human blood plasma biomarker discovery via improved in-depth analysis of the low-abundant N-Glycoproteome. GlycoBioTec 2023, Berlin, Germany (2023)
Hoffmann, M.; Pioch, M.; Pralow, A.; Hennig, R.; Kottler, R.; Reichl, U.; Rapp, E.: The Fine Art of Destruction: A Guide to In-Depth Glycoproteomic Analyses – Exploiting the Diagnostic Potential of Fragment Ions. Proteomics 18 (24), e1800282 (2018)
Hoffmann, M.; Marx, K.; Reichl, U.; Wuhrer, M.; Rapp, E.: Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins. Molecular and Cellular Proteomics 15 (2), pp. 624 - 641 (2016)
Pioch, M.; Hoffmann, M.; Pralow, A.; Reichl, U.; Rapp, E.: GlyXtoolMS: An Open-Source Pipeline for Semiautomated Analysis of Glycopeptide Mass Spectrometry Data. Analytical Chemistry 90 (20), pp. 11908 - 11916 (2018)
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