Glycosylation is one of the most common post-translational modifications in mammalian biology, yet the glycans it adds to proteins remain surprisingly difficult to measure. A new study in Analyst reports a combined strategy that raises the mass spectrometry signal of N-linked glycans by an average of 9-fold in solution and 27-fold in tissue, with individual peaks climbing past 100-fold. The work, by Eisenberg and Muddiman, pairs ammonium fluoride electrospray doping with an electrospray diverter on the IR-MALDESI imaging platform. For laboratories that depend on sensitive, spatially resolved glycan analysis, the method offers a practical route to detect many glycans that previously fell below the limit of detection. This is good news for Glycomics, where the difference between a visible peak and a missed one often determines whether a biological pattern can be mapped at all.
Why N-Linked Glycans Are Hard to See
More than half of mammalian proteins carry glycans, and these sugar chains participate in cell signaling, adhesion, trafficking, and protein stability. Abnormal or disrupted glycosylation is linked to cancer, autoimmune disease, and infection, which is why the field tracks it so closely. Despite this importance, glycans are notoriously quiet in a mass spectrometer.
N-linked glycans share a conserved pentasaccharide core and are added only at specific amino acid motifs. They can be prepared using a Enzymatic Release of N-Glycans that cleaves them from the protein backbone, after which they become accessible to detection. The core problem is ionization. In ambient and imaging workflows, neutral glycans ionize poorly, and the ions that do form are easily overwhelmed by background ions drawn in from the open atmosphere. These background signals cause ion suppression, pushing true glycan peaks below the noise.
This challenge is acute in mass spectrometry imaging (MSI), where hundreds to thousands of ions are mapped across a tissue section at once. Ambient ionization sources such as DESI, SESI, APCI, DART, MALDI, and IR-MALDESI generate ions without a vacuum, reducing sample preparation and preserving tissue context. They belong to the broader family of Mass Spectrometry-Based Glycan Profiling, but because they operate in air they also collect ions unrelated to the sample. For researchers who study glycosylation in tissue, that contamination is the difference between seeing a glycan and missing it.
Two Independent Routes to a Stronger Signal
The authors built on two previously reported tricks, each of which raises signal through a different mechanism.
Ammonium Fluoride Doping Deprotonates Neutral Glycans
Adding a small amount of ammonium fluoride (NH4F) to the electrospray solvent changes how neutral analytes charge. The electronegative fluoride ion captures protons from neutral glycans, generating deprotonated [M−H]− ions that are far easier to detect. In earlier IR-MALDESI work, this doping raised glycan signal by up to 4-fold and improved detection frequency by about 30 percent, without altering the structures themselves.
The Electrospray Diverter Blocks Background Ions
The second trick attacks the background directly. Many trapping mass analyzers, including Orbitraps, must move ions through a flight path before they are caught in the trap. During that transit, unrelated background ions can slip in and fill the trap, suppressing the analytes of interest. An electrospray diverter intercepts this process: it opens to admit sample-related ions right after the laser fires, then closes to divert excess background ions away from the inlet. Earlier demonstrations showed it could cut background by up to 4-fold and lift sample signal by up to an order of magnitude. The effect is strongest for large m/z species such as glycans and proteins, which take the longest to reach the trap.
Combining the Two: ESI-DD
Because these two approaches work through orthogonal mechanisms, they can be applied together without interference. The combined workflow, called ESI-DD, uses an electrospray solvent containing 350 µM NH4F together with the diverter timed to the laser event.
Fig. 1 Schematic outlining the process of using ESI-DD for an experiment. (Eisenberg, et al. 2026)
The synergy is the key result. Ammonium fluoride doping lifts signal broadly, improving both background and analyte ions, while the diverter discards the background so the ion trap fills with sample-related ions. Acting together, they produce gains far larger than either alone. Where each technique individually delivered 2-fold to 10-fold improvements, the combination pushed many glycans up by two orders of magnitude.
In Solution: Bovine Fetuin N-Glycans
To test the approach, the team first analyzed N-linked glycans cleaved from bovine fetuin, a single well-characterized glycoprotein and a frequent reference for an N-Glycan Profiling. Using a 384-well plate and the ESI-DD setup, they detected 21 glycoforms from that one protein.
Compared with standard IR-MALDESI conditions, the ESI-DD abundance showed an average 9-fold enhancement, with a maximum of 33-fold for individual glycans. Low-abundance glycans were lifted past the limit of detection so their isotopic peaks became visible, while already-strong glycans gained visibility and detection frequency. Importantly, the enhancement was statistically independent of the glycan adduct, the presence of fucose or sialic acid monosaccharides, and the glycan type. That uniformity suggests the technique is robust and largely agnostic to analyte structure, which matters for any workflow that aims to compare many structures at once.
In Tissue: Human Kidney Sections
The more demanding test was imaging. Formalin-fixed, paraffin-embedded human kidney tissue was cut and prepared for MSI, then each section was split so that one half was analyzed under control conditions and the other half under ESI-DD. Comparing intra-tissue halves removes biological variability between samples, so any difference must come from the method.
In tissue, the gains were even larger. Among 15 detected glycans, enhancement ranged from 2-fold to 130-fold, averaging 27-fold. Because the control and ESI-DD data came from opposite halves of the same tissue, the increase is clearly driven by the method, not by biology. Many of these glycans were below the limit of detection without ESI-DD and would simply have been missed. The tissue also revealed doubly and triply deprotonated glycans and chlorine adducts from the sample buffer, and Sialic Acid Analysis comparisons confirmed that sialic acids appeared without any derivatization, with structures consistent with the sialic acid rule.
Fig. 2 Box and whisker plots evaluating the enhancement factor caused by ESI-DD, comparing the effect caused by various factors. (Eisenberg, et al. 2026)
Why This Matters for Glycomics
The practical value is straightforward. Researchers using trapping-based instruments can realize large signal gains with minimal changes to setup or hardware. For N-glycan analysis in particular, the ability to pull low-abundance species above the detection threshold means richer spatial maps of glycosylation and a better chance of catching disease-associated changes. The authors note that the next step is using the platform to map N-linked glycans across tissues and between tissue conditions, exactly the kind of comparative work that drives biomarker discovery.
Although the tissue glycans needed no derivatization in this study, a Glycan Derivation remains valuable when linkage-specific information is required, and the released structures can be handed to a Glycomic Characterization for full confirmation. Together these steps turn a stronger raw signal into validated, reproducible measurements that other laboratories can build on.
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Reference
- Eisenberg, S. M., et al. (2026). Glycan signal enhancement by ammonium fluoride doping and electrospray diverting. Analyst. DOI: 10.1039/d6an00090h.
