Probing the Molecules of Life

Dr Peter Nemes is fascinated by the societal impacts of technology and its pivotal role in driving scientific discoveries. An associate professor in the Department of Chemistry and Biochemistry at the University of Maryland, his research drives innovation in bioanalytical instruments and their applications.

These next-generation mass spectrometry (MS) technologies are capable of measuring molecules that have important implications in developing biological systems. They can be utilized to study single cells or even whole embryos, helping us to understand the complex molecular systems that underpin cell and neurodevelopmental biology, cell fate and disease.

Technology Networks recently interviewed Nemes to learn how his lab is "digging in a goldmine of discoveries" in developmental biology, what the future of MS-based analysis might hold and the challenges that come with probing the molecules of life that only exist for a matter of minutes.

Molly Campbell (MC): Why is it important to study neuroscience and developmental biology from a proteomics/ metabolomics perspective, particularly at the single-cell level?

Peter Nemes (PN): During healthy development, molecules called transcripts, proteins and metabolites must be produced at the correct concentration, at the correct time and the correct place. Decades of biological research has focused on the production and developmental significance of important transcripts – mostly – and some proteins. However, mounting evidence from recent studies reveals complex correlations between transcripts and proteins in developing systems, thus necessitating the direct analysis of proteins and metabolites. The challenge has been the detection of these molecules in sufficient sensitivity in the miniscule amounts of materials contained by single-cells and neurons. For example, MS, the number one detection technology for these molecules, requires the pooling of thousands-to-millions of cells, thus losing information on each cell.

Only recently have my lab and others built bioanalytical instruments with sufficient sensitivity to detect metabolites and proteins in single cells and neurons. By measuring the production of these molecules at a single-cell resolution, my lab has uncovered previously unknown differences between stem cells that reproducibly form neural and epidermal tissues, and signaling canters in the vertebrate frog embryo, as well as single neurons in the mouse central nervous system.

Our functional biological studies on these molecules discovered metabolites capable of reprogramming cell fates, perturbing signaling and even affecting behavior by the whole organism. These outcomes would not have been revealed had only transcripts been measured in the single cells, because the transcripts in our developing embryo do not necessarily correlate with proteins and, especially, metabolites. This example demonstrates how integration of information on the molecular status of cells from chemistry, with functional insight from cell/developmental biology and neuroscience, opens exciting new possibilities to understand molecular programs that cells execute during states of health and disease.

MC: Can you talk to us about the key challenges with regards to the MS-based study of proteins/metabolites during development?

PN: MS-based proteomics/metabolomics faces many challenges in cell/developmental biology and neuroscience – that is also precisely where our chemistry background comes into play in my lab. Most of these challenges call for specialized tools and methodologies from instrumental bioanalytical chemistry. For example, miniscule amounts of proteins and metabolites must be collected from small cells that may or may not be embedded in complex tissues or even entire organisms such as the three-dimensionally complex (frog) embryo. As if that was not difficult enough for us, development itself imposes strict time scales on our experiments. The cells that we study during neurodevelopmental processes only exist for ~15–20 minutes before the embryo develops to the next stage. That means that the members of my group only have a few minutes to collect the cells before they are, well, long “gone”, meaning we would have to start the work all over again.

These collected materials must then be processed without losing much of the material on surfaces, pipette tips and vials, etc. Molecules in the resulting samples must be separated, efficiently ionized and then identified and quantified by MS instruments.

Complex biological environments yield thousands to hundreds of thousands of signals, demanding exquisite power in resolution, specificity and sensitivity. These molecules are also present at broad concentration ranges, usually covering a 7-to-10-decade dynamic range. My laboratory (among others) has developed specialized bioanalytical mass spectrometry platforms and protocols to address these key challenges.

MC: Your laboratory takes an interdisciplinary approach to solve the above challenges by developing next-generation mass spectrometry technologies. Can you discuss some of the recent platforms and methodologies that you have developed?

PN: The students and postdocs have worked very hard in my lab to develop several technologies and methodologies to address these bioanalytical challenges. I am indebted by their hard work and dedication.

Dr Rosemary Onjiko, Dr Camille Lombard-Banek and Dr Kellen Delaney, as well as PhD graduate students Erika Portero and Sam B Choi, have developed specialized ways to manually dissect identified cells/neurons or probe their chemical constituents directly in the live embryo or in brain tissues. They have precision fabricated microcapillaries of scalable sizes to collect proteins and metabolites from single cells and neurons of varying sizes in “lightning speeds”. In less than five seconds, we are now able to collect the cells!

The team have also custom-built capillary electrophoresis instruments to separate proteins, peptides and metabolites in high resolution, and our home-built CE electrospray ionization interfaces convert these molecules in high efficiency.

In parallel, Choi and Zheng have introduced intelligent strategies and software packages based on artificial intelligence to detect, sequence, identify and quantify these biomolecules in ultra-high sensitivity. With low attomole-to-zeptomole lower limits of detection, we can detect ~2,000+ different proteins, including many low-abundance transcription factors, and ~100+ different metabolites, including several neurotransmitter molecules, by sampling only a portion of the single cell’s or neuron’s content.

Further, Leena Pade and Jie Li, as well as other members of the group, are developing exciting new ways to decipher molecular changes in single differentiating cells and their clones during development. Stay tuned for upcoming publications from our group to learn about these exciting progresses!

Developing A Single-Neuron Protein Mass Analyzer. Video credit: UMD Sciences.

MC: What are some of the key insights that you have gained when applying such platforms/methodologies for cell/ developmental biology research?

PN: “We are digging in a goldmine of discoveries", members of my group and I often joke. The home-built ultrasensitive MS platforms have allowed my group to uncover previously unknown molecular differences between single cells that reproducibly differentiate to different types of tissues. We have also used these tools to characterize the proteomic content of single-neurons and single-tissue nuclei in the mouse nervous system. By conducting functional experiments based on cell fate tracking, imaging and microinjections, we have discovered metabolites that are able to reprogram epidermally fated cells to become neural tissue and vice versa.

Portero most recently discovered molecular communication between neighboring cells in the embryo, which contribute to the patterning of the embryonic body. These findings on the metabolically driven patterning of the embryonic emerge as a new chapter in cell/developmental biology and neuroscience. We also have multiple projects in which we study molecular mechanisms underlying neural communication and memory, auditory perception and impairment and various abnormalities of development, including cancer.

MC: What benefits are there to taking an interdisciplinary approach to research in this context?

PN: By integrating chemistry, software development, biology and neuroscience, we have a unique opportunity to better understand how chemistry drives biology and vice versa at the cellular level, thus supporting the development of efficient therapeutics.

While the science is exciting, it has been humbling and extremely rewarding to be able to train the next generation of scientists through my lab’s interdisciplinary program. Several of the trainees in the lab are from traditionally underrepresented minorities in science, technology, engineering, and mathematics (STEM) fields, thus serving as encouragement and outstanding role models for others. By the end of tenure in my lab, these talented young professionals develop a natural aptitude to converse across biology and chemistry. That skillset allows them to tackle current chemistry-biology questions more efficiently and ask new questions that have important implications for society at large. I do not need a crystal ball to know that the future of science will be bright in the hands of these talented scientists.

I do not need a crystal ball to know that the future of science will be bright in the hands of these talented scientists – Nemes.

MC: Can you discuss the challenges that remain in single-cell proteomics and metabolomics, both for applications in neuroscience research and in other fields of research?  

PN: There are so many challenges, but fortunately, even more solutions are being put forward thanks to the hard work of many colleagues from many countries and continents.

Without the possibility to amplify the whole proteome and metabolome, single-cell proteomics and metabolomics hinges on the detection sensitivity afforded by MS. Whilst my lab and others have made great strides, it will be important to continue the development of efficient ways to sample, process, separate, ionize and detect these molecules by mass spectrometers, as well as to bring these developments to other scientists.

In parallel, it will be essential to advance software tools to extract important information from the multidimensional mass spectrometry data resulting from these studies. Fortunately, these interconnected steps can be addressed in independent laboratories, thus amplifying developmental efforts to hopefully bring single-cell proteomics and single-cell metabolomics from a few specialized labs, like mine, to the “masses.”

MC: What aspect of proteomics/ metabolomics research currently excites you the most, and what are your personal aspirations in this space for the future? 

PN: I am thrilled to see the importance of science and scientists in bettering, and driving, our shared future. Let us just think about the impact of light microscope, mass MS and gene editing. Just three centuries ago, microscopist-microbiologist Antonie van Leeuwenhoek developed the compound microscope, discovering mystical “animalcules”, or microorganisms, as we call them today. His microscopy work pioneered microbiology and set a motion in stage in many areas of biology, including modern cell biology, neuroscience, bacteriology and virology.

Only two decades ago, Profs. John Fenn and Koichi Tanaka shared the Nobel Prize in Chemistry for their pioneering works in biological MS, paving the way to modern proteomics and metabolomics. This technology has become indispensable in basic and applied biology, including clinical science. In 2020, Profs. Emmanuelle Charpentier and Jennifer Doudna received the Nobel Prize in Chemistry for developing CRISPR-Cas9, genetic scissors that have revolutionized the investigation of many cell biological questions and raised a potential for treating certain types of genetic diseases. I am intrigued by the many research possibilities that the integration of these tools (and other approaches) will afford to make discoveries, with MS taking a central stage in molecular analysis.

Single-cell MS has the potential to help revolutionize many areas of biology and health research. This technology enables the detection of proteins and metabolites and their quantification without requiring functional probes (no antibodies necessary). It can be made scalable to the size of single cells and cells located in complex environments. Single-cell mass spectrometry can also be integrated with technologies of cell biology and neuroscience, thus driving new innovations. For example, in our lab, the integration of microscopy, tools of cell/developmental biology and neuroscience, and the technologies of MS-based proteomics and metabolomics led to the discovery of cell fate altering metabolites and metabolic cell-to-cell communication.

Other labs are also making exciting advances in the field of single-cell MS, including new technologies to speed up single-cell proteomics (e.g., nanoPOTS MS) and correlate single-cell transcriptomics with single-cell proteomics data (e.g., SCoPE MS). How fascinating would it be to learn to use this type of information from single-cell mass spectrometry to grow skin, liver kidney, etc. on demand without a risk of organ rejection? Or to develop next-generation treatments tailoring to the chemistry, via the “ome”, of a patient!?  

Dr Peter Nemes was speaking to Molly Campbell, Science Writer for Technology Networks.