Image: © VBCF-Ludwig Schedl

Manuel Matzinger, Senior Core Scientist and Deputy Head of the Proteomic Technology Hub at the Campus Vienna Biocenter, is pushing the boundaries of single-cell proteomics by combining the latest MS technologies with innovative workflows. Now, his most exciting challenge involves lowering input levels down to single cells to investigate cellular heterogeneity in tissues, during disease development or embryogenesis.

The path to proteomics

“To understand how the world functions in detail and what happens upon changing one part of the puzzle always fascinated me in my childhood,” Manuel recalls. This early curiosity led him first to a chemistry-focused high school, and later to a PhD investigating the behaviour of the transcription factor Nrf2, which becomes active upon cellular stress, on a molecular level.

It was during this time that mass spectrometry emerged as the ideal analytical tool for his research. “Mass spectrometry turned out to be the ideal analytical tool to get a comprehensive and unbiased idea of how this transcription factor functions and which partner proteins are involved,” he explains.

The single-cell revolution

As Manuel’s career progressed, he found himself drawn deeper into the world of proteomics, particularly the challenge of single-cell analysis. “Right now, lowering input levels down to single cells to investigate cellular heterogeneity in tissues, during disease development or embryogenesis is among the most exciting challenges in my daily life as researcher,” he shares.

The journey to making single-cell proteomics a reality was far from easy. “When I started to think about single cell proteomics, it was an interesting technical challenge but nothing that was really feasible,” Manuel continued. The team had to overcome numerous hurdles, from handling tiny volumes to dealing with evaporation and finding ways to improve sensitivity by multiplexing and using of carrier proteins. The latter, however, introduced entirely new challenges, like ratio compression.

“Back then, we were happy to identify a couple hundred proteins.”

The “aha” moment

The breakthrough came when they finally managed to generate single-cell samples as a one-pot preparation without ever touching the sample with a pipette tip. “When we finally managed to… quantify ~1000 proteins/cell, this was a huge milestone for us. It was the first time I thought – well, it’s possible!” Manuel exclaims.

This moment marked a turning point, not just for their research, but for the entire field of proteomics. Technological improvements in both hardware and software followed rapidly, catapulting their results to ~5,000 quantified proteins per cell. Among the most significant technological improvements, Manuel lists having a new generation of MS instruments in his hands, using shorter gradients for better throughput, and switching his chromatography to Aurora Series for their true-zero dead volume and sharpest possible peaks.

Pushing the boundaries

In their most recent paper, the team explored the capabilities of Thermo Scientific’s new Astral mass analyser in combination with IonOpticks columns and their One-Pot Workflow. “To the best of our knowledge, we were the first to assess this at single cell input levels using a two-proteome mix,” Manuel notes.

One of the highlights of this study was the ability to find heterogeneity even within a presumably homogeneous system of cultured and untreated cells.

Find the full study here.

Image: © VBCF-Ludwig Schedl

Navigating challenges in proteomics

Like any scientific endeavour, Manuel’s journey has been filled with challenges. “For a scientist, it seems quite common that most ideas fail while a small fraction leads to success,” he reflects. One recent issue his team grappled with is maintaining proper control of the false discovery rate (FDR) in their analyses.

“This reflects one of the biggest challenges in proteomics, especially single-cell proteomics,” Manuel explains. “The control of FDR has been discussed for decades, but at the same time we’re seeing a race for high numbers in the community.”

This race for high numbers has led to some concerning trends. Modern data analysis software often employs various techniques to boost identification numbers, such as matching spectra across files. While these methods can increase the number of identified proteins, they can also lead to decreased confidence in the results.

“In some cases, this might lead to peptides given as true positive which have only very few or no clearly annotated fragment ion for identification,” he cautions. “This results in lowered confidence, and those identifications have to be confirmed with proper care.”

To address this issue in their own work, the team implemented additional checks and balances. They performed entrapment searches against a database of a different organism to estimate the FDR and control the output from their proteomic search software. When they found that default settings led to a higher-than-expected FDR, they adjusted their approach, implementing more stringent search settings based on findings from colleagues facing similar challenges.

Looking to the future

Asked about the most exciting recent developments in proteomics, Manuel describes the potential of spatially resolved proteomics at the level of few cells to subcellular resolutions, particularly for clinical research and diagnostics.

“This could mean that cellular subpopulations of a tumour could be identified and mapped back to their position within the patient or organ,” he explains. “In combination with AI-based training datasets, this information could be used to predict at a very early stage which part of the tissue is malignant or likely transitioning to be malignant and metastasise.”

“Such information at an early stage could save lives and improve quality of life, which is the most important overall goal of our research efforts.”

Where do you fall in the debate on FDR? Have your own developments in proteomics you think are exciting? Email us at [email protected] and tell us a bit about yourself for a chance to be featured in a future Community Newsletter.