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Fixing the Huge Leak at the Ion Source in Mass Spec

A graphic of proteins within a cell.
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Mass spectrometry (MS) is considered the gold standard method in modern proteomics, but it isn’t without its limitations.


Ionization is an essential step in MS analysis, and there are a variety of methods available for ionization depending on the sample type and research goal. Electrospray ionization (ESI) has become an increasingly popular technique for the study of molecules in complex biological samples, but it has a major limitation: sample loss.


“MS is an amazingly powerful technique that can easily resolve the mass differences between the amino acids, and it employs detectors that can measure single ions. The problem lies with electrospray ionization (ESI), the standard method used to introduce ions into the mass spectrometer,” Dr. Derek Stein, professor emeritus of physics at Brown University, told Technology Networks. 


The conventional ESI technique typically transfers only ~one percent of the analyte into the mass spectrometer. It’s a major drawback for researchers in the field, and particularly for Stein, whose lab is working to develop a single-molecule protein sequencing technique that combines MS and nanopore technology. “Our single-molecule protein sequencing concept will not work if 99% of the amino acids to be identified are lost by the ion source,” he explained.


Over the last decade, Stein and colleagues have been working hard to address this limitation, culminating in the development of a new advancement in MS technology – a nanopore ion source – published in Nature Communications.

Circumventing the Coulomb explosions that results in high sample loss

“To understand what makes our nanopore ion source innovative, it helps to understand the problem that it overcomes in a bit more detail,” Stein said.


“The conventional ESI technique for producing vapor phase ions involves squeezing a liquid sample through a small tube while applying a voltage to the liquid. The tube ejects charged droplets into an environment filled with a gas (called a ‘background’ gas) that helps the solvent in the droplets evaporate. As the droplets shrink, the electric charge they contain gets squeezed closer together until the electric forces eventually grow so strong that they cause a droplet to explode into smaller, daughter droplets,” he continued.

This event is known as a Coulomb explosion. A string of Coulomb explosions spreads out generations of daughter droplets into a wide plume, which results in a high loss of analyte because the droplets are effectively flying around in many different directions.


"This low transfer efficiency is a fundamental problem, especially when one wants to measure small samples like single cells or single molecules,” Stein said.

“But there is hope! The very last phase of ESI doesn’t occur until the charged droplets shrink down to just a few nanometers in size. At that scale, Coulomb forces drive ions directly out of the liquid and into the vacuum by a process called ion evaporation. These are the vapor phase ions that a mass spectrometer ultimately measures.”

Stein and colleagues had an idea. What if they could shrink the ion source down to a few tens of nanometers across, and put it directly in front of the mass filter?


“By applying a strong voltage to the nanoscale tip, ions can be pulled directly from the liquid meniscus into the vapor phase. This does not involve the release of charged droplets, so it circumvents the Coulomb explosions that usually result in so much sample loss,” Stein said. “Instead, the nanopore ion source delivers ions directly into the vapor phase by the mechanism of ion evaporation.”


The researchers developed a tiny capillary that has a ~30 nm diameter orifice, roughly 600 times smaller than the needle used in conventional ESI.


Making the capillary is a relatively easy process, Stein described: “We pull on the ends of a thin quartz tube while a laser heats the middle of the tube. The laser softens the quartz, and it stretches out like the cheese on a slice of pizza as you pull it away. The quartz tube thins until it snaps in the middle. With practice, the laser heating and pulling forces can be tuned to give a nanoscale orifice nearly every time.”


The Nature Communications paper is as a proof-of-concept study using the new transfer method. Stein and colleagues successfully measured the mass spectra of 16 amino acids, post-translationally modified versions of glutathione and the peptide angiotensin II, demonstrating that their technology matched the capabilities of existing methods, but with a reduced amount of sample lost in the process.

A diagram of the new nanotube for nanopore ion source MS.

The new nanotube also has the unique ability to transfer ions that are dissolved in water directly into the vacuum of mass spectrometers. Credit: Photo provided by Nicholas Drachman.

Developing an efficient technique for single-molecule protein sequencing

Biological samples are precious material, particularly in clinical research that requires patient samples. Reducing the amount of starting sample required for proteomic analysis is an ongoing goal for the field, and one that could be tackled by nanopore ion source technology.


“By fixing the huge leak at the ion source, which loses about 99% of the interesting proteins at present, it should be possible to work with about one one-hundredth the amount of sample,” Stein said.


Fixing the leak will also be critical for achieving Stein’s goal of developing an efficient technique for single-molecule protein sequencing: “MS is a robust method for identifying amino acids, but single-molecule protein sequencing will require a new kind of ion source that can transmit amino acids without significant loss. And unlike DNA or RNA, which can be easily manipulated using enzymes that read or replicate them in a processive manner, nature does not provide equivalent enzymes for proteins,” he said.


“This lack of natural, processive mechanisms for protein manipulation further complicates single-molecule sequencing, requiring labor-intensive techniques like Edman degradation or tandem MS techniques that require large ensembles of molecules,” Stein continued.


By delivering vapor phase ions of amino acids directly to the high vacuum region of a mass spectrometer, the nanopore ion source provides a new way to avoid that sample loss. Fragmenting a protein down and using the nanopore ion source to deliver its amino acids to a mass spectrometer in sequential order could be a new pathway to single-molecule protein sequencing. “A key challenge is to measure the ordering of molecular components, which should be encoded in the arrival times of ions at the detector because, without any background gas to pass through, the ions follow deterministic trajectories,” Stein said.


“We are working on ways to use light to fragment a protein into separate amino acids while it is still in the liquid inside the ion source. This is the next step on the roadmap of our single-molecule protein sequencing concept,” Stein said.


The research team is also redesigning the nanopore ion source, such that it can be evaluated using commercial MS instruments, which is an important step for potential commercialization.  


Professor Derek Stein was speaking to Molly Coddington, Senior Science Writer and News Team Lead at Technology Networks.


Reference: Drachman N, Lepoitevin M, Szapary H, Wiener B, Maulbetsch W, Stein D. Nanopore ion sources deliver individual ions of amino acids and peptides directly into high vacuum. Nat Comms. 2024;15(1):7709. doi: 10.1038/s41467-024-51455-x


About the interviewee


Professor Derek Stein joined the Physics Department at Brown following postdoctoral work at the Kavli Institute of Nanoscience at the Delft University of Technology in the Netherlands. He obtained his PhD in applied physics from Harvard University and his BSc in physics from McGill University. Stein has broad research interests, which include nanotechnology and its applications to the study of single biomolecules. Stein is also the founder and CEO of a startup company that develops advanced moisture control materials for buildings.