Native mass spectrometry (MS) is increasingly used to study proteins and protein complexes under conditions that preserve aspects of their native structure, but its reliance on volatile buffers remains a practical limitation. Many biologically relevant systems are sensitive to changes in ionic strength, buffer composition, and other solution conditions, meaning that conventional sample preparation can alter the conformational states or interactions researchers aim to preserve.
Rather than adapting proteins to the demands of native MS, Emily Byrd and colleagues asked whether nanopipette emitters could adapt the ionization process instead. Their recent study in Analytical Chemistry used sub-100 nm emitters and ion mobility mass spectrometry to probe α-synuclein directly in biochemical buffers, capturing how changes in buffer composition and ionic strength reshape its conformational ensemble. Complementary benchmarking across proteins and instrument platforms assessed whether those benefits could translate beyond a single model system.
Here, Byrd discusses the analytical challenges that still limit routine native MS, the development and cross-platform potential of nanopipette emitters, and how the approach could expand access to more complex and biologically relevant samples.
What are the main practical barriers that still limit routine native MS measurements?
One of the biggest barriers is sample compatibility. Native mass spectrometry analyzes proteins in volatile buffers such as ammonium acetate, but many biologically relevant systems are not particularly stable under these conditions. Physiological salts, phosphate buffers, detergents, lipids, cofactors and complex matrices can all lead to adduction, ion suppression, peak broadening and reduced sensitivity. This creates a trade-off between applying enough source activation to achieve efficient desolvation and applying so much that native protein complexes begin to dissociate.
Another challenge is that different protein classes can behave very differently. Small globular proteins, metalloproteins, large protein complexes, intrinsically disordered proteins and membrane assemblies all have different requirements for preserving structure and interactions. Different MS platforms also have different source geometries, transmission efficiencies and activation requirements, meaning a workflow that performs well on one instrument or protein class may not transfer easily to another.
Making native MS more routine therefore requires approaches that are robust, consume very little sample and are compatible with a broader range of sample conditions and MS platforms.
What initially motivated your team to investigate nanopipette emitters for native MS?
The motivation came from a very practical problem: we wanted to analyze proteins and protein assemblies under conditions that better preserve their biology. However, standard native MS often requires extensive buffer exchange into ammonium acetate or other sample preparation steps. While that can be manageable for stable purified proteins, it can perturb weak interactions, remove cofactors, alter conformational ensembles, and make low-abundance species difficult to detect.
The collaboration came about during my postdoctoral research at the University of Leeds. Professor Paolo Actis had been working with nanopipettes as single-molecule sensors for many years, and his team had developed reproducible protocols for fabricating and characterizing sub-100 nm nanopipettes. Paolo had always been interested in our MS approaches, so one day we decided to give his nanopipettes a try, without knowing whether they would actually be applicable to native MS.
Nanopipettes presented a promising alternative. Because of their remarkably small internal diameter, they produce much smaller droplets than conventional nano-electrospray emitters. The idea was that smaller droplets should carry less salt into the gas phase, improve desolvation and reduce nonspecific adduction, while still maintaining gentle ionization. Lane Baker’s group at Texas A&M and Evan Williams’ group at UC Berkeley had demonstrated the benefits of smaller-diameter emitters several years earlier, so we wanted to explore whether this could be applied robustly across different proteins and native MS platforms.
What began as a fairly exploratory experiment ultimately developed into the work we completed in Joseph Loo and Rachel Ogorzalek Loo’s laboratory at UCLA, supported by a research grant I received from the British Mass Spectrometry Society. By combining the Actis group’s extensive expertise in nanopipette fabrication and characterization with our native MS workflows, we could then test whether these emitters were robust and broadly applicable across different proteins and instrument platforms.
At that point, we realized a quick Friday afternoon experiment might just have become something more legitimate.
Could you explain, in a nutshell, how these nanopipette emitters work?
Conventional nano-electrospray emitters have openings in the micrometre range that are often pulled closed, requiring manual opening using metal tweezers under a light microscope. This can introduce variability, as each emitter ends up with a slightly different diameter. They work very well for many native MS experiments, but they still generate droplets that can contain substantial solvent and salt. Our nanopipettes, by contrast, have openings below 50 nm and operate at very low flow rates, generating much smaller initial droplets.
Conventional emitters are typically fabricated from borosilicate glass using a filament puller. Nanopipettes, however, are made from quartz glass and pulled using a laser puller. Quartz has a much higher melting temperature than borosilicate, so the temperatures needed to produce the emitters are not compatible with conventional filament pullers. Together, the higher temperature and rapid pulling produce the much finer tip diameters characteristic of the nanopipettes.
That difference in droplet size is important. Smaller droplets carry less salt and can support gentler transfer of proteins and complexes into the gas phase. In practice, this means we often observe cleaner spectra, fewer salt adducts, improved signal-to-noise and better preservation of native-like features of analytes measured in buffers such as PBS. This allows us to improve the electrospray process itself, rather than relying on prior sample clean-up.
Overall, the approach reduces the workload involved in sample preparation and clean-up. It also mitigates issues such as sample loss from buffer exchange columns and aggregation when proteins are not happy in ammonium acetate.
What did the cross-platform benchmarking reveal?
The main finding was that the nanopipettes were transferable across platforms and vendors, and across a broad mass range, from small proteins such as myoglobin (17 kDa) and carbonic anhydrase (29 kDa) to much larger protein complexes such as GroEL (800 kDa).
For myoglobin, we were able to detect clean native spectra across a concentration series, including at very low concentrations, while retaining the holo, or heme-bound, form without prior desalting. With carbonic anhydrase, we observed preservation of the zinc-bound metalloproteoform alongside reduced nonspecific salt adduction. We also saw strong performance for mid- and high-mass assemblies, including protein complexes in the hundreds of kilodaltons, as well as compatibility with different Orbitrap- and Q-TOF-based native MS platforms.
The broader message is that these nanopipettes are not limited to one specific protein, experiment or instrument platform. They offer a practical, cross-platform approach for improving native MS spectral quality, reducing adduct burden and lowering sample consumption across multiple protein classes.
Were there any results that surprised you?
What surprised me the most was that the approach worked well for large protein complexes. Take β-galactosidase, for example: its largest diameter is around 18 nm, while our nanopipettes measure around 20–30 nm. That suggests we may be approaching something close to single-ion exit from the emitters. We like to envision them queuing up to leave the tip.
We were also surprised by how effectively the emitters performed across very different instruments. Native MS source conditions can be quite instrument-specific, so I anticipated that we would need to optimize the setup more extensively. Instead, the nanopipettes were relatively easy to translate between platforms once we understood the key operating parameters, including substantially lower electrospray voltages (down to 0.2 kV).
I was also excited by the preservation of labile features, such as the heme-bound form of myoglobin, the zinc-bound form of carbonic anhydrase and the noncovalent oligomeric states of complexes, alongside cleaner spectra with fewer adducts. That combination is important because improving desolvation can sometimes come at the cost of disrupting native-like features. Here, the nanopipettes helped to reduce adduction while still maintaining cofactor binding and noncovalent interactions.
Another exciting result was their performance with more complex samples, including lysate-based measurements. That brings us closer to using native MS not only for purified standards, but for biologically relevant matrices where sample amount and buffer or salt composition are major limitations.
What were the biggest technical or analytical challenges your team faced, and how did you overcome them?
One of the technical challenges with nanopipettes is slight variability between laser pullers. This means that different laboratories can produce emitters with somewhat different internal diameters, even when using similar fabrication protocols.
We addressed this by combining fabrication with emitter characterization, led by the Actis lab. Scanning electron microscopy imaging helped us assess tip morphology and diameter, while electrical measurements provided an orthogonal way to estimate diameter and check batch consistency. In day-to-day experiments, we also found that careful sample loading to avoid bubbles, as well as spinning the loaded nanopipettes down, both helped significantly.
Analytically, the challenge was making fair comparisons across instruments and emitter types. To do so, we used a panel of proteins spanning different masses and behaviors, and assessed spectral quality across several criteria: including signal, adduction, charge-state resolution, cofactor preservation and complex detection, rather than relying on a single metric.
How could this approach change the way researchers use native MS for more complex biological samples?
I think the biggest impact is that nanopipettes could make native MS compatible with a wider range of physiologically relevant buffers, including PBS, sodium and potassium phosphate, and Tris-HCl. That could make many more proteins and biological samples accessible to native MS without first forcing them into conditions that may alter their native state. Instead of focusing on how to make analytes happy in a volatile buffer, we can start asking, “can we adapt the ionization process to preserve more of the original biology?”
For protein complexes, this could help maintain weak or salt-dependent interactions. For proteoforms, cleaner spectra and reduced adduction make it easier to detect small mass shifts, PTMs, cofactors, oligomeric states and ligand binding. For complex samples such as lysates or biofluids, reduced sample consumption and improved salt tolerance could help move native MS closer to direct analysis of real biological mixtures rather than relying on bottom-up proteomics approaches alone.
This does not remove the need for careful sample preparation or orthogonal validation. But it does provide a simpler front-end strategy that can be combined with complementary techniques such as ion mobility, native top-down MS, charge detection/direct mass technology and other structural MS methods.
Where do you see the greatest opportunities for this approach, and what still needs to happen for wider adoption?
I see the greatest potential for impact in applications where sample amount is limited, buffer exchange is undesirable, or the biology is highly sensitive to solution conditions. Potential applications include intrinsically disordered proteins, amyloid assemblies, metalloproteins, membrane-associated systems, nucleic acids and clinical biofluids.
I am particularly excited about combining nanopipette native MS with ion mobility and top-down fragmentation. This could enable us to observe intact proteoforms and conformational ensembles directly in biofluids such as cerebrospinal fluid, with potential applications in neurodegenerative disease biomarker discovery. This will be a major next step in my work with Anouk Rijs (Vrije Universiteit Amsterdam) and Joseph Loo, in collaboration with Paolo Actis.
For wider adoption, we first need standardization. That includes reproducible pulling protocols across different tip diameters, practical quality-control metrics, guidance on emitter handling and loading, and consistent reporting of emitter dimensions and source conditions. We also need broader benchmarking across laboratories, instruments and sample classes.
If we can make the workflow robust and accessible, I think nanopipettes could become a very useful addition to the native MS toolbox – especially for researchers trying to bridge the gap between purified model systems and truly native biological samples.
Emily Byrd is a Marie Skłodowska-Curie Global Postdoctoral Fellow at Vrije Universiteit Amsterdam, the Netherlands.
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