Unprecedented protein quantification in single cells using advanced proteomics

In this interview, first author Julia Bubis and corresponding author Manuel Matzinger, both scientists of the Proteomics Tech Hub, discuss the innovative method, its applications, and the challenges and rewards of the project.
What’s the idea behind this new method?
Manuel: Single-cell proteomics can be compared to wanting to know the different elements of a smoothie. Imagine you take a fruit salad, blend it into a smoothie, and taste it. Sure, it might taste great, but it's just the average flavor of all the fruits mixed together. If there was only one strawberry in the mix, you'd never know—it’s lost in the blend. To find out if there was one strawberry, you’d need to analyze the fruits one by one. That’s exactly what single-cell proteomics does–instead of analyzing millions of cells all at once, it studies one cell at a time, allowing us to find rare or unique cell types hidden within the larger group.
How do you analyze proteins at the single-cell level?
Julia: To look inside a cell and quantify its proteins, we start by breaking the cell open. Then, we use molecular scissors—or enzymes—to cut the proteins into smaller pieces called peptides. These peptides are ionized and sent into the mass spectrometer, which measures each peptide’s mass-to-charge ratio, giving us a kind of fingerprint that helps us reconstruct and identify each protein. Mass spectrometry allows us to pick out individual proteins in a mix, determine how many there are, and even see differences between seemingly identical cells. The new workflow we have developed allows us to do this at the single cell level, and with great precision.
What makes this technical development important?
Julia: Our method tackles a key limitation in studying proteins: unlike RNA, proteins can’t be amplified with PCR, so we need super-sensitive methods to detect them at the single-cell level.
Manuel: To perform single-cell proteomics, we needed to develop a workflow that kept as much material as possible from the very start of sample preparation. So, we optimized every step—sample preparation, chromatography, and the mass spectrometry itself —using AstralTM, a brand-new instrument from Thermo Fisher Scientific. We were allowed to test Astral directly at the factory, which was a game changer.
How does your method achieve this level of precision and resolution?
Julia: Achieving this level of precision came down to two main factors. First, both Manuel and I are part of the Proteomics Tech Hub, where we specialize in method development. We’ve worked extensively on creating an optimized sample preparation protocol and fine-tuning parameters across all stages of the experiment. Second, we had access to the recently released Astral mass spectrometer. Before Astral was developed, there were two leading types of mass spectrometers: time-of-flight analyzers, which are extremely fast but provide lower resolution; and Orbitrap detectors, which are super precise but slower. The new Astral analyzer is a brilliant piece of engineering that combines the best of both worlds: it uses a clever design to reflect ions multiple times inside a compact space, creating an effective flight path of over 30 meters and minimizing ion loss, which boosts resolution, sensitivity, and speed all at once. Joining forces allowed us to detect up to 5,300 proteins per single cell, an unprecedented level of proteome depth.
How did this project come together?
Manuel: Thermo Scientific invited us to try out the Astral, and we spent a week testing the new instrument. We were blown away by its performance, and that’s when the project really took off. We quickly started flashing out ideas to test the limits of the machine and its applications. The biggest surprise? We ended up publishing results from what was initially just a demo session!
What applications does your new method have?
Julia: The method we have developed is very versatile and has a lot of exciting applications, especially for understanding the fundamentals mechanisms of cellular biology. It’s particularly useful for systems that are small and naturally heterogeneous, like the blastoid, a model of the early-stage embryo with just 100 cells and three cell lineages. Traditional methods can’t study these effectively because they average everything out, but single-cell proteomics lets us see the biology of each cell population. In addition, focusing on proteins —the real workhorses of the cell—and their modifications gives us a clearer, more detailed picture of what’s happening inside the cell at the molecular level.
Manuel: Single-cell proteomics is a fairly new branch of proteomics that’s adding a fresh dimension to biological research. It lets us study proteins at the individual cell level, complementing what we already know from DNA and RNA studies to give a more complete picture of what happens inside each cell. We show it can detect heterogeneity even in what seems like a uniform cell population, like looking at 60 cells from the same line and spotting differences in their proteomes. We started with this simple model to test the method, and we showed that it provides sufficient resolution to analyze individual cells effectively, even at this level.
What was the most rewarding aspect of working on this project?
Manuel: Well, the opportunity for great collaboration. Our team at the Proteomics Tech Hub focused on method development, which is our specialty at the Vienna BioCenter. We partnered with Thermo Fisher Scientific to access their cutting-edge instrument before it was even on the market. We also worked with Nicolas Rivron’s lab at IMBA, who shared their blastoid models and their expertise on embryonic development.
Julia: For me, the most exciting moment was realizing that we could uncover heterogeneity within a seemingly uniform cell population—without needing any additional stimuli. The depth of the proteome coverage and the precision of quantification allowed us to hypothesize about the cell cycle phases: whether a cell had just divided or was about to. This level of detail was unprecedented and highlighted just how powerful our method is for detecting subtle differences between cells of the same type—something older techniques simply couldn’t achieve. Seeing how proteins—the real drivers of cellular behavior—can reveal these insights felt like a game-changer, and I’m excited to see what we can do next.
Further Information
Read the full paper here
About Manuel Matzinger
Manuel Matzinger pursued his studies in Chemistry at the University of Vienna and the University of Bergen, Norway. He earned his PhD in pharmaceutical chemistry, focusing on transcription factors activated by oxidative stress. In 2020, he joined the Proteomics Tech Hub at the Institute of Molecular Pathology (IMP) as a postdoctoral researcher. During his time at IMP, Manuel co-conceptualized and co-authored several successful grant applications, including FWF grants and an FFG infrastructure grant, which facilitated the acquisition of state-of-the-art mass spectrometers. Additionally, he plays a key role in organizing the annual European Symposium on Single Cell Proteomics. His research centers on developing advanced analytical techniques in crosslinking mass spectrometry and ultra-low-input proteomics. Since November 2024, Manuel has served as a Senior Core Scientist at the Proteomics Technology Hub of the Vienna BioCenter.
About Julia Bubis
Julia Bubis earned her Ph.D. in Molecular and Chemical Physics from the Moscow Institute of Physics and Technology in 2022, where she specialized in developing high-throughput liquid chromatography-mass spectrometry (LC-MS) methods and quantitation strategies. She joined the Proteomics Technology Hub at the Vienna BioCenter as a postdoctoral researcher. Julia serves as the principal investigator for the "Single-cell Proteomics of Human Blastoid" project, funded by the Austrian Science Fund's ESPRIT program, focusing on advancing liquid chromatography-mass spectrometry techniques for single-cell proteomics and data analysis to get better understanding of mechanisms of early-stage embryo development using blastocyst-like structures. Her achievements have been recognized with multiple awards, including the Seal of Excellence from the Marie Curie Fellowship in 2023 and the Best Poster Award at the Single Cell Proteomics Conference in Boston in 2024.