New workflow enhances detection of low-abundant glycoproteins in blood plasma

A recent study published in Engineering presents a novel approach to analyzing the low-abundant N-glycoproteome in human blood plasma (HBP), which could significantly advance the discovery of plasma biomarkers.

Protein glycosylation plays a crucial role in clinical diagnostics and biopharmaceuticals. However, current N-glycoproteomic methods face challenges such as incorrect identifications, difficulties in detecting rare and modified N-glycans, and insufficient coverage, especially in complex samples like blood plasma. To address these issues, the research team developed an innovative N-glycoproteomic workflow.

The workflow begins with the depletion of the top 14 high-abundant blood plasma proteins (HAP) and a fractionation strategy. This is followed by tryptic digestion and glycopeptide enrichment. The samples are then analyzed using high-resolution mass spectrometry with stepped collision fragmentation (HCD.step and HCD.low). A new decision tree procedure is incorporated for data validation.

The results of the study are promising. The sample preparation workflow extends the detection range of glycoproteins in blood plasma. It can detect glycoproteins with concentrations as low as 6.31 pg·mL⁻¹, expanding the range by five orders of magnitude compared to direct plasma analysis. The data analysis workflow enables the reliable differentiation of ambiguous N-glycan structures. For example, it can distinguish between antenna and core fucosylation, as well as identify modified and rare N-glycans such as sulfated and glucuronidated ones.

In total, 1929 N-glycopeptides and 942 N-glycosites derived from 805 human middle- to low-abundant glycoproteins were identified. The researchers also detected sulfated and phosphorylated N-glycopeptides in common HBP glycoproteins. Moreover, they discovered three rare N-glycan building blocks with masses of 176.0314, 245.0524, and 259.0672 Da.

This new workflow not only improves our understanding of protein glycosylation but also has the potential to be applied in various fields. It can be used to explore biomarker candidates in HBP, evaluate biotherapeutic proteins, and study biological models. Although the study has limitations, such as the relatively long measurement time of the instrument, the findings provide valuable insights for future glycoproteomic research. Overall, this research offers a new avenue for the in-depth analysis of HBP glycoproteins and biomarker discovery.

The paper "New Avenues for Human Blood Plasma Biomarker Discovery via Improved In-Depth Analysis of the Low-Abundant N-glycoproteome," is authored by Frania J. Zuniga-Banuelos, Marcus Hoffmann, Udo Reichl, Erdmann Rapp.