Proteomics Technologies and Applications

SPONSORED BY Proteomics Technologies and Applications Recent Applications of Proteomics Single-Cell Proteomics: Mass Spec vs Single- Molecule Sequencing How Collaboration and Curiosity Make for a Successful Scientist Credit: iStock Recent Applications of Proteomics 4 Single-Cell Proteomics: Mass Spec vs Single-Molecule Sequencing 8 Advances in Proteomics & Metabolomics 2023 10 Infographic: Applications of Mass Spectrometry 12 News Roundup: Protein Biomarkers and Disease 13 Infographic: Western Blotting Troubleshotting 15 How Collaboration and Curiosity Make for a Successful Scientist 16 Advancing Antigen Discovery With Microfluidics Automation for Sparse Samples 19 Detecting Cancer From a Droplet of Blood 21 Contents 2 TECHNOLOGYNETWORKS.COM 4 TECHNOLOGYNETWORKS.COM Credit: iStock. The advent of “omics” has changed the scale of life sciences research. While transcriptomics provides a useful overview of global gene expression, proteomics attempts to provide a comprehensive insight into the expression, structure and function of the entire set of proteins encoded by an organism at a given time.1,2 Technological and methodological advances in proteomics workflows have contributed to a linear increase in proteomic investigations across different fields, including medical research, drug discovery, microbiology and plant biology (Figure 1). This listicle outlines basic concepts of proteomic workflows and lists some of the latest applications across different research disciplines. METHODS AND TECHNIQUES The proteomic era has recently blossomed thanks to the advances in mass spectrometry (MS) technologies and data analysis tools. The analysis of proteins can be grouped into three main types:3 Recent Applications of Proteomics Mariana Gil, PhD Proteomics 1992 1997 2002 2007 2012 2017 2022 Year Number of publication Figure 1: Growth in the number of publications using proteomic workflows. Data sourced from PubMed. Credit: Mariana Gil 5 TECHNOLOGYNETWORKS.COM Proteomics Credit: iStock. TechnologyNetworks Cell or Tissue Data Analysis Peptide Generation Protein Protein Extraction Protein Digestion Protein Extraction Protein Separation Cell or Tissue MS Detection and Analysis Proteins MS Detection and Analysis Data Analysis TOPDOWN BOTTOMUP • Expression proteomics measuring changes in the levels of protein expression under different conditions • Structural proteomics studying the three-dimensional structure of proteins and how they change in response to different conditions • Functional proteomics aiming to understand proteins’ function and their interactions. Typically, proteomics workflows include four steps:3,4 1. Sample preparation methods depend on the sample and the goal of the experiment. This is a critical step as its accuracy can determine the success of the whole workflow. 2. Separation can be achieved using antibody-based methods (i.e., western blotting and enzyme-linked immunosorbent assay (ELISA)), gel-based methods (i.e., SDS-PAGE and 2D-DIGE) or chromatography-based approaches (i.e., affinity chromatography, size exclusion chromatography (SEC), ion-exchange chromatography (IEXC) and liquid chromatography (LC)). 3. Identification of proteins can be achieved using MS, Edman sequencing or protein microarrays. 4. Data analysis is typically performed using sequence database searching software (e.g., Sequent, Mascot, Comet and Tandem). This general workflow can be applied to top-down and bottom- up approaches (Figure 2). In top-down proteomics, the proteins in a sample are first separated before being individually characterized.3,5 In contrast, in bottom-up proteomics, all the proteins in a sample are first digested using trypsin and the resulting mixture of peptides is then analyzed.3,5 LATEST APPLICATIONS Proteomics has revolutionized medical research and enables researchers to identify novel biomarkers for the diagnosis and treatment of different diseases, including cancer, neurodegenerative disorders and infectious diseases.3 It is also extensively used in drug discovery to understand the protein targets of newly developed drugs and to monitor their effect on the protein levels.3 YetNumber of publications of proteomics go beyond human health. For example, it can be applied to agriculture,6 animal husbandry,7 environmental science,8 food science,9 forensics10 and astrobiology.11 Some of the latest studies across different disciplines are explored below. Biomedical research Autoimmune encephalitis (AE) includes a group of non-infectious inflammatory conditions of the central nervous system caused by an imbalanced immune response. In a recent study, researchers used proteomics to understand the complex pathophysiological mechanisms of this disease. Dr. Saskia Räuber and colleagues compared the cerebrospinal flu- Figure 2: Top-down and bottom-up approaches for proteomics. 6 TECHNOLOGYNETWORKS.COM Proteomics id protein profile of different types of AE patients and control patients. They found that AE patients have a dysregulation of proteins involved in inflammatory processes, synaptic transmission, synaptogenesis, brain connectivity and neurodegeneration. Moreover, patients with different AE subtypes showed distinct protein profiles which may facilitate future identification of disease-specific biomarkers.12 Drug discovery Small molecule therapeutics are used to treat a broad range of diseases. However, their exact mechanism of action (MoA) is not always known. Researchers have now developed a high-throughput screening method that uses quantitative proteomics to measure proteome-level changes induced by drugs. Using this method, Dr. Dylan Mitchell and his team screened a library of 875 small molecule drugs and quantified more than 8,900 proteins. They then built a proteome fingerprint database that can be used to elucidate the MoA and off-target effects of several compounds.13 The routine implementation of this type of method during early stages of drug development is expected to increase efficiency of the drug discovery pipeline. Food industry Proteomics is also extensively used to characterize food composition, quality and safety. Sturgeon meat is a rich source of highly digestible protein. However, protein oxidation during sturgeon fillets processing decreases the quality of the meat.14 Low temperature vacuum heating (LTVH) is an easy-to-use processing method that seems to improve the processing quality of sturgeon meat.14 In a recent study, Dr. Dan-dan Jiang and colleagues used a proteomic approach to characterize protein oxidation of sturgeon meat after LTVH treatment. The analysis of 733 proteins revealed that LTVH produced mild oxidation and may provide a theoretical basis to improve the processing of aquatic products.15 Plant biology and agriculture Proteomics applied to plant biology is a powerful tool that can help to develop new plant-based products, improve plant health and increase crop yields. Soybean is one of the most important economic crops worldwide, providing an abundant source of plant protein and oil for humans and livestock. The lysine 2-hydroxyisobutyrylation (Khib) is a post-translational modification (PTM) described in different organisms including several plant species.16 Dr. Wei Zhao and colleagues recently used proteomics to identify Khib -modified proteins in soybean leaves for the first time. The analysis identified 4,251 Khib sites in 1,532 proteins involved in a wide range of cellular processes (biosynthesis, central carbon metabolism and photosynthesis).17 This data is useful to gain insight into the regulatory mechanisms of Khib and may help to improve soybean yields. Animal husbandry and welfare Animal welfare is a major concern in animal husbandry as it can affect food quality and safety as well as determine consumers’ preferences. Road transportation, for example, is a common practice in the livestock industry, affecting both animal welfare and meat quality.18,19 Proteomics analysis can be applied to this area of research to support animal welfare strategies. For example, Dr. Alessio Di Luca and colleagues used a label-free LC-MS proteomic workflow to compare the proteome of pigs after short or long road transportation. The analysis of 1,464 proteins revealed that 66 were expressed differentially in the 2 populations of animals.20 The identification of these stress-related biomarkers may be used to improve animal transportation conditions and ensure animal welfare. Environmental science Proteomics has also proved helpful in monitoring the effects of pollution and climate change on ecosystems. For example, the Mediterranean Sea is currently one of the major hotspots of microplastics (MPs) pollution. In a recently published article, Dr. Carola Murano and her team explored the effect of MPs on the sea urchin immune cells proteome. They found that MPs exposure altered the protein profile in a concentration-dependent manner. MPs appear to increase the expression of metabolite interconversion enzymes involved in cellular processes, indicating a severe alteration of the cellular metabolic pathways.21 This type of study provides new insights on the mode of action of pollutants at the molecular and cellular level. Forensics Proteome analysis is also useful in forensics to identify individuals, assess evidence and solve crimes. Recent studies show that it can be used to determine death due to rattlesnake envenomization22 and abusive head trauma.23 Moreover, post-mortem analysis of the bone proteome has proved useful to estimate the age-at-death and post-mortem interval.24,25 Space exploration Although less common, proteomic analyses are also used in space research aiming to support long-term space missions and the establishment of life on other planets. NASA’s planned mission to Mars will present several health challenges to astronauts, including the exposure to ~350 mSv of galactic cosmic radiation during each year of the mission. Experiments in rodents suggest that exposure to this type 7 TECHNOLOGYNETWORKS.COM Proteomics of space radiation impairs a variety of cognitive functions. In a recent study, Dr. Evagelia Laiakis and her team studied the proteomic profile of the medial prefrontal cortex of rats exposed to space radiation. They identified several alterations in pathways and proteins that correlate with the rats’ cognitive performance.26 In the future, these biomarkers can potentially be targeted for development of appropriate countermeasures. CONCLUSION Proteomics is currently in the limelight and open to a myriad of applications across the life sciences, from biomarker discovery to space exploration. But the field is still evolving. The future may see the rise of several promising technologies for the high-throughput single-molecule sequencing of proteins that are currently hampered by sensitivity, throughput or cost.5 References 1. Wilkins MR, et al. Biotechnol Genet Eng Rev. 1996;13:19–50. 2. Beynon RJ. Brief Funct Genom. 2005;3(4):382–390. 3. Al-Amrani S, et al. World J Biol Chem. 2021;12(5):57–69. 4. Aslam B, et al. J Chromatogr Sci. 2017;55(2):182–196. 5. Timp W, Timp G. Sci Adv. 2020;6(2):eaax8978. 6. Eldakak M, et al. Front Plant Sci. 2013;4:35. 7. Chakraborty D, et al. Front Genet. 2022;13:774113. 8. Lacerda CMR, Reardon KF. Brief Func Genom. 2009;8(1):75–87. 9. Carrera M. Foods. 2021;10(11):2538. 10. Parker GJ, et al. Forensic Sci Int Genet. 2021;54:102529. 11. Somogyi Á, et al. Int J Mol Sci. 2016;17(4):439. 12. Räuber S, et al. J Autoimmun. 2023;135:102985. 13. Mitchell DC, et al. Nat Biotechnol. 2023;41(6):845–857. 14. Shen SK, et al. Food Chem X. 2022;15:100389. 15. Jiang DD, et al. J Sci Food Agric. 2023;103(6):2858–2866. 16. Dai L, et al. Nat Chem Biol. 2014;10(5):365–370. 17. Zhao W, et al. BMC Plant Biol. 2023;23(1):23. 18. Lammens V, et al. Meat Sci. 2007;75(3):381–387. 19. Shen QW, et al. Meat Sci. 2006;74(2):388–395. 20. Di Luca A, et al. PLoS One. 2022;17(11):e0277950. 21. Murano C, et al. Environ Pollut. 2023;320:121062. 22. Gallagher T, et al. J Forensic Sci. 2023;68(2):711–715. 23. Wiskott K, et al. Proteomics. 2023;23(3-4):e2200078. 24. Bonicelli A, et al. elife. 2022;11:e83658. 25. Gent L, et al. J Proteomics. 2023;271:104754. 26. Laiakis EC, et al. Front Physiol. 2022;13:971282. ww 8 TECHNOLOGYNETWORKS.COM Proteomics Credit: iStock Single-Cell Proteomics: Mass Spec vs Single-Molecule Sequencing Molly Campbell Unlike bulk proteomics, which analyzes average protein expression across a group of cells, single-cell proteomics explores the heterogeneity and diversity of single cells. These deeper insights offer a new understanding of cell biology, cellular responses to stimuli and how complex signaling pathways contribute to the cell’s function as a system. When studying human health and disease, this field is set to have a profound impact on how we diagnose and treat illnesses in personalized medicine. Medical fields such as cancer research are already benefiting from single-cell proteomics, which can shed light on why certain cells within a tumor respond to a specific therapy, while others may not, for example. The single-cell proteomics toolbox continues to evolve, with novel workflows combining a variety of analytical tools being published frequently. The dominant setup for most experiments involves the use of mass spectrometry (MS) in some configuration. Lately, there has been excitement surrounding single-molecule sequencing and its possible applications in proteomics. Dr. Ryan Kelly is an associate professor in the Department of Chemistry and Biochemistry at Brigham Young University. His research centers around the development of new technological solutions for ultrasensitive biochemical analyses, including single-cell profiling and high-resolution proteome imaging. Technology Networks interviewed Kelly to hear his thoughts on the single-cell proteomics landscape and the potential impact of single-molecule analysis. Q: For readers that are unfamiliar, how does single- cell MS proteomics differ from single-molecule protein sequencing? A: Thousands of copies of a given peptide are needed to make a confident measurement by MS, and this is what we do with single-cell proteomics. The non-MS-based single-molecule protein sequencing approaches (based on nanopores, etc.) measure individual copies of each peptide, but these have not been used to measure peptides or proteins in single cells yet. The fields of single-cell proteomics and single-molecule protein sequencing will converge at some point, but they haven’t yet. Q: Many MS vendors are creating instruments that support single-cell MS proteomics. Why would single-cell protein sequencing be advantageous, given the advancements in the MS space? Could this be adopted in complement to single-cell MS proteomics, or do you envision this could be a technology that competes with MS? A: Single-cell MS is more “ready for prime time”, while single-molecule sequencing is generally still in a proof-ofconcept stage. I suspect that as single-molecule sequencing matures, there will be areas of complementarity and areas of competition. For example, single-molecule sequencing 9 TECHNOLOGYNETWORKS.COM Proteomics technologies by their very nature detect single molecules, while MS requires a few thousand or tens of thousands of copies to make a measurement. So sequencing approaches could potentially fill in the gap on ultra-trace-level proteins, particularly if those low-abundance species can be separated from the higher abundance ones. Q: What are the key challenges faced by both single-cell MS proteomics and protein sequencing? A: Both technologies have dynamic range and measurement throughput limitations. More effective sample preparation and separations can also benefit both fields. Q: You have previously said that you believe “rumors of mass spectrometry’s demise have been greatly exaggerated”. Can you expand on this viewpoint? A: The non-MS proteomics technologies tend to be purpose- built for a given application or have a predefined panel of target analytes. If you want to study something outside of that range, you’re out of luck. In contrast, the same mass spectrometer can be used to study protein digests, intact proteins, post-translational modifications, lipids, carbohydrates, etc., from all biological systems. It’s this versatility that will allow MS to fill in the gaps even as other emerging technologies mature. Q: How do you see the proteomics research field evolving over the coming years? A: If single-cell proteomics continues to advance to where it becomes very high throughput and very sensitive, it could become the default mode of proteome profiling. A similar trend is already happening with transcriptomics. Measuring populations of cells one at a time provides both that single- cell granularity and population-level information at the same time. Dr. Ryan Kelly was speaking to Molly Campbell, Senior Science Writer for Technology Networks. This popular online event will once again bring together leading scientists in the field as they highlight the latest advancements in translational proteomics and metabolomics. Presentations will cover areas ranging from uncovering the molecular mechanisms of disease pathology, to the latest technological developments in diagnostics and treatment. Take a front row seat at our FREE Online Symposium to hear more on: • Cutting-edge technologies and collaborative strategies in proteomics • Innovations in separations and data acquisition for single-cell proteomics • Advances in plasma lipidomics • The evolution of next-generation protein sequencing • Uncovering the latest fluid biomarker breakthroughs advancing disease understanding REGISTER NOW Technology Networks frequently host educational webinars that span the breadth of our scientific coverage, from coronavirus to cannabis. Previous hosts include Nobel prize laureate Professor Jennifer Doudna and members of the team who pioneered the Oxford/AstraZeneca COVID-19 vaccine. You can view all of our latest webinars HERE. Advances in Proteomics & Metabolomics 2023 View our lineup of expert speakers Credit: iStock “extraordinary science” Extraordinary scienceCoined by philosopher and science historian Thomas Kuhn,refers to discoveries that go beyond current knowledge, challenging understandings and paving the way for scientific revolutions.The SCIEX clinical diagnostic portfolio is For In Vitro Diagnostic Use. Rx Only. Product(s) not available in all countries. For information on availability, please contact your local sales representative or refer to www.sciex.com/diagnostics. All other products are For Research Use Only. Not for use in Diagnostic Procedures. Trademarks and/or registered trademarks mentioned herein, including associated logos, are the property of AB Sciex Pte. Ltd. or their respective owners in the United States and/or certain other countries (see www.sciex.com/trademarks). © 2022 DH Tech. Dev. Pte. Ltd. MKT-29296-AWATCH NOWBe inspired by the extraordinary science from proteomics researchers using SCIEX ZenoTOF 7600 system.Visit:sciex.com/extraordinary-science In the clinic, MS is becoming a vital tool, able to detect and quantitate trace levels of known and unknown targets in complex samples. Advancements in areas such as ionization have helped to create growing applications of MS in the clinic, including: Clinical diagnostics PRIMARY STRUCTURE Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS can identify bacterial species rapidly and has been used to detect antimicrobial resistance, informing treatment plans MS-based proteomics is a powerful tool in disease biomarker detection, spanning conditions from type 1 diabetes to chronic kidney disease By studying protein expression profiles by MS alongside genomic profiles, clinicians are able to tailor treatment plans to best suit the responses of individuals MS imaging (MSI) offers great insights on the localization of biomolecules. For example, in neuroscience, it has been used to image neurotransmitters in brain slices MICROBIOLOGICAL TESTING BIOMARKER DETECTION PERSONALIZED ONCOLOGY BIOMOLECULE LOCALIZATION MS is a powerful and essential tool in the field of omics, specifically proteomics, metabolomics, lipidomics and glycomics. Among others, it provides insights for: Omics Investigating fundamental biology Identifying potential drug candidates Understanding and predicting disease Process refinement in producing desirable products Diagnosis of infectious and non-infectious disease Nutritional research DOWNLOAD TO LEARN MORE 13 TECHNOLOGYNETWORKS.COM Proteomics Credit: iStock News Roundup: Protein Biomarkers and Disease Kate Robinson Biomarkers can play a valuable role in understanding the pathology of disease as well as helping to drive the development of diagnostics and therapeutics. From cancer to neurodegenerative disease, this roundup explores recent protein biomarker discoveries. New Protein Biomarker for Alzheimer’s Disease Discovered In a study published in Genome Medicine, a team of researchers from the Max Delbrück Center (MDC) discovered a novel protein, called Arl8b, that builds up in the brains of Alzheimer’s patients. In Alzheimer’s disease, amyloid-beta peptides clump together in the brain to form plaques that lead to inflammation and eventually cause neuronal cell death. In the study, the team genetically modified mice to have five mutations that occur in individuals with familial Alzheimer’s disease. They found that while amyloid-beta plaques developed in the mice’s brains, Arl8b built up too. Unlike brain tissue, cerebrospinal fluid is easily accessible for diagnostic studies. “This means Arl8b is an interesting candidate for a diagnostic marker,” said Dr. Annett Böddrich, lead author of the study. “Our work shows that proteomic research can provide crucial information for identifying disease mechanisms and markers, and thereby move research forward. Also, this doesn’t just apply to Alzheimer’s; it’s also relevant to other complex neurodegenerative diseases such as Parkinson’s and Huntington’s,” said Professor Erich Wanker, head of the Proteomics and Molecular Mechanisms of Neurodegenerative Diseases Lab at the MDC. Reference: Boeddrich A, et al. Genome Med. 2023;15(1):50. Protein Biomarkers Help To Predict Islet Autoimmunity Researchers at the Department of Energy’s Pacific Northwest National Laboratory (PNNL) have identified a set of altered proteins that predict islet autoimmunity, a precursor for Type 1 diabetes. Currently, there is no way to determine if or when either islet autoimmunity or diabetes will occur in genetically predisposed individuals. In the study, published in Cell Reports Medicine, PNNL scientists analyzed blood plasma samples of almost 1,000 children from birth up to the age of 6. The researchers identified a set of 83 proteins whose combination of changes predicted which children went on to develop either islet autoimmunity or Type 1 diabetes. “What’s exciting about this work is that it opens the door to detecting autoimmunity earlier than we can right now,” said Dr. Thomas Metz, senior scientist, 14 TECHNOLOGYNETWORKS.COM Proteomics PNNL. “This gives us an opportunity to learn more about what causes the immune system to turn on the body. This could help us tease out and understand the mechanisms at play in the development of diabetes better than we do currently and provide potential targets for intervention.” Reference: Nakayasu ES, et al. Cell Rep Med. 2023;4(7). New Urine Tests May One Day Detect Early Bladder Cancer Researchers from the University of Houston (UH) have discovered new biomarkers for early detection of bladder cancer. The current gold standard for bladder cancer diagnosis, cystoscopy, is associated with complications including pain, urinary tract infection and blood in the urine. It also often lacks the sensitivity to correctly identify the disease. This study, published in BMC Medicine, demonstrates the first and largest use of comprehensive aptamer-based proteomic screening of urine samples from bladder cancer patients to analyze the expression of over 1,300 proteins. Using this screening method, the researchers were able to identify 21 urine proteins discriminating bladder cancer from urology clinic controls. One such protein, urine D-dimer, displayed the highest accuracy and sensitivity. “Using aptamer-based screening, we analyzed the expression of 1,317 proteins in the urine of bladder cancer patients and found that D-dimer - a protein fragment from the breakdown of a blood clot - may have a role in the initial diagnosis or detection of cancer recurrence,” said Dr. Chandra Mohan, Hugh Roy and Lillie Cranz Cullen Endowed Professor at UH. Reference: Vanarsa K, et al. BMC Med. 2023;21(1):133. Risk Biomarkers for Chronic Graft-Versus-Host Disease Identified In a study published in Journal of Clinical Investigation, researchers have identified three risk biomarkers that could be measured long before a doctor would be able to make a clinical diagnosis of chronic graft-versus-host-disease (GVHD). Patients undergoing allogeneic hematopoietic cell transplantation face the possibility of GVHD, where the donated cells begin to attack the patient’s own healthy cells. Severe GVHD can be quite debilitating and is a major cause of death for patients. The team of researchers identified three biomarkers that could be measured at 90 days after transplantation. Two of the biomarkers, MMP3 and DKK3, are associated with fibrosis, the hardening or scarring of tissue due to over-repair. The third marker, CXCL9, is a type of protein that attracts particular immune cells into the organs under attack. “Current diagnosis is based on clinical signs that may be confirmed by invasive biopsy of skin and appendages, mouth, female genitalia, esophagus, lungs, and connective tissues. Unfortunately, these signs often reveal late-stage fibrotic lesions, as opposed to early lesions that may be more amenable to treatment,” the authors wrote. Reference: Logan BR, et al. J Clin Invest. 2023;133(15). New Prognostic Biomarker for Heart Failure Identified According to a study published in New England Journal of Medicine Evidence, levels of endotrophin in the bloodstream can be used to predict outcomes in patients with a common form of heart failure. In heart failure with preserved ejection fraction (HFpEF), the heart loses pumping efficiency because its main pumping muscle becomes too stiff to relax sufficiently between pumping actions. This stiffening involves fibrosis, in which normal muscle is replaced by scar-like tissue. Animal studies suggest that endotrophin, a fragment released during the formation of type VI collagen, is related to the development of both fibrosis and metabolic dysfunction. Both processes are thought to be important in HFpEF. In the study, the researchers analyzed endotrophin levels in blood samples taken from 205 HFpEF patients at the outset of a previous clinical trial. They split the patients into three tiers according to their endotrophin levels, and compared how they fared in the trial. Over a four-year follow-up period, patients in the highest tier had a several fold increased risk of having a heart attack, being hospitalized for the management of heart failure or dying from any cardiovascular cause. “In addition to helping us gauge the risks faced by HFpEF patients, endotrophin could give us important clues to the biological processes underlying poor outcomes in this form of heart failure—and might even be a target for treatment,” said Dr. Julio Chirinos, associate professor of Cardiovascular Medicine at the University of Pennsylvania. Reference: Chirinos JA, et al. NEJM Evidence. 2022;1(10). Why a western blot ? • Western blotting is a molecular biology technique that allows the presence (or absence), size and abundance of a specific protein to be determined, even within a complex mixture of proteins from cells or tissues. • This means that the technique can be used amongst other things to verify the success of gene editing or modification experiments using protein expression, investigate disease and detect tagged proteins. • Western blotting is similar to Southern and northern blotting, however these techniques are used to detect DNA sequences and RNA sequences, respectively. • This infographic will take you through the multistep western blotting process and give some tips to avoid poor results. and tricks Western Blot Troubleshooting When loading samples onto the gel, it’s good practice to make a note of what is in each well. Before you begin, make sure the electrodes are the correct way round - if not, you could lose your sample in the buffer. Mixture of proteins Gel Before SDS-PAGE After SDS-PAGE Medium molecular weight protein Low molecular weight protein Download to learn more 16 TECHNOLOGYNETWORKS.COM Proteomics Credit: iStock. Dr. Birgit Schilling. How Collaboration and Curiosity Make for a Successful Scientist Molly Campbell Dr. Birgit Schilling studies the molecular mechanisms that underlie aging processes and is particularly passionate about research that could lead to novel therapeutic interventions for human aging or disease. At the Buck Institute for Research on Aging in Novato near San Francisco, CA, she leads her laboratory in the use of modern proteomics technologies – such as dataindependent acquisition – to fulfill these research goals. Key project examples from her lab include investigating the dynamic role of post-translational modifications (PTMs) during cell signaling, specifically in the context of metabolic diseases, neurodegenerative diseases, cancer and aging. Dr. Schilling studied chemistry at the University of Hamburg in Germany and she also studied as an undergraduate student in Southampton. After completing her PhD in Germany, she started a postdoctoral fellowship in 1998 at the University of California San Francisco (UCSF). She has worked at the Buck Institute since 2000, where she is also the director of the Mass Spectrometry Technology Center. Dr. Schilling has a long-standing scientific track-record in developing mass spectrometry (MS)-based methodologies for quantitatively analyzing complex samples. She is incredibly well respected in her field and has collaborated with many scientists across the globe. Technology Networks champions diversity in science and embraces the opportunity to learn from women in science about their journey – the challenges they may have faced, how their research focuses evolved and the advice they may offer young scientists eager to follow in their footsteps. We recently had the pleasure of interviewing Dr. Schilling, who talked about the “beauty in the chemistry that generates life”, why science careers can be hard and the importance of knowing when to change direction. Dr. Birgit Schilling is a professor and director of the Mass Spectrometry Technology Center at the Buck Institute for Research on Aging. 17 TECHNOLOGYNETWORKS.COM Proteomics Q: What inspired you to pursue a career in science? A: I was always fascinated by the natural world around me and how one could use science to try to explain natural phenomena. I studied chemistry, and I really liked the logic and elemental understanding of what is around us. Most of all, I really love the multi-disciplinary aspects of science, and how chemistry, biology, physics and medicine all inform each other and often yield wonderful collaborations between scientists from different backgrounds. Science is highly collaborative, which I cherish, and those collaborations can be national and international. What intrigued me when I studied chemistry is how nature has brought forward fascinating chemical structures and molecules that “operate” and manifest life and often show “healing” power. Quite a few pharmaceutical drugs are based on natural products, and in some cases these structures are synthesized and further optimized to generate therapeutics that become medical interventions for disease treatments – this is also referred to as “natural product chemistry”. There is a beauty in the chemistry that generates life. Q: Why did you decide to focus your research on aging, specifically? A: The aging process of life – how an organism develops, grows and ages – is a fascinating field of research. We are interested in “healthy aging”, meaning to extend what we call “health span” so that humans have a longer span of health throughout their life. Interestingly, aging is closely related with many age-related diseases, so a better understanding of aging will help to tackle age-related diseases. Aging is also something that we all will face, but understanding the complexity of this biological process is important to implement interventions – in lifestyle (exercise/diet) or pharmaceutically. Connecting aging mechanisms with, for example, neurodegenerative brain diseases, such as Alzheimer’s disease – but also other devastating diseases, such as cancer – is interesting from a scientific standpoint and will contribute to the development of further interventions. Q: As a woman in science, have you faced any barriers in your career journey? A: That is an interesting question. I have always been a “strong” person. When I studied chemistry in my college years, there were so few female students, it was really surprising. But what counted was the science and I could usually easily connect and interact with anybody. We were good friends supporting each other – it did not matter whether we were men or women. So that was not a problem. Throughout my career, I was focused on my scientific work and would try hard to showcase the skills and passion that I had. I wanted to be “measured” based on that – the good science I could contribute and the fact I was a good collaborator. I did not get too much pushback, and when I did, that was just not the direction I would pursue, I would re-orient myself. There are so many opportunities, if one thing does not work out, I would look for other directions, or if I cared a lot for something that led to me facing obstacles, I would try to really show what I can do and convince those around me with my good work. I found great supporters that would help me move forward – but I also put in a large amount of work to make my case! I have found many scientists do support each other when they collaborate well, and when they see how valuable the work of the other person is. Q: What qualities and values do you think makes for a successful scientist? A: Curiosity, persistency, enthusiasm and a fascination with learning something new every single day – imagine that! What is also important is having a keen eye for understanding a scientific observation: many people may observe the same thing, but it is the right interpretation of an experimental outcome that sometimes reveals the most interesting scientific results. Being a scientist is not always easy because sometimes experiments are challenging, but when things go well, it is so rewarding. Finding the right collaborators, the right projects to engage in, showing persistency but also knowing when to change direction are incredibly important. I think being a team player is key and being open to embracing the joy of collaborating. Also, recognizing how technology can help scientists is very important, in addition to finding the “right” place to do your fun science. Something to ask yourself if you are thinking of being a scientist is: do you love nature – plants, animals, humans, microbes or other? Are you interested in understanding how life works? We can use science to help, for example, in human diseases, or for other scientific purposes. An overall appreciation for the complexity of life is a good quality for scientists to have. 18 TECHNOLOGYNETWORKS.COM Proteomics Q: Can you talk about women in science that have inspired or supported you? A: This is an interesting question – until I was >30 years old, there were no women in science who had inspired me. My history teacher in high school, who is a woman, really inspired me with her strength and knowledge and other interests outside of her school work (travel and photography). In my college years, there were no women professors in Chemistry (in Germany at the time). When I was an early postdoctoral fellow, I met Dr. Catherine (Cathy) Costello, who is an amazing scientist and professor at Boston University. She was so kind as to give me a ride from an Asilomar Mass Spectrometry conference back to San Francisco Airport. During the journey, we talked about many things, both within and outside of science. That was so important to me – to experience how somebody can be such a highly respected and successful scientist, admired by many (myself included!), but also be a kind person with so much generosity on a personal level. Other women scientists who have really supported me are Dr. Jennifer Van Eyk at Cedars-Sinai in Los Angeles and Dr. Ileana Cristea at Princeton. At the Buck Institute, I am so lucky to work with Dr. Judith (Judy) Campisi and Dr. Lisa Ellerby, who both are dear colleagues. We do great science together and we support each other. I also know a lot of male scientists who I admire greatly, and who have helped me a lot in my career. I usually look at the person (man or woman) – and then I connect with them as a person and a scientist. It is the connection that generates a great scientific colleague – somebody who I support and who may support me. Q: What advice would you give to someone that wishes to pursue a career in science? A: Follow your dreams. If science is what you like to do – then go for it. It is hard at times – but also greatly rewarding. Dr. Birgit Schilling was speaking to Molly Campbell, Senior Science Writer for Technology Networks. 19 TECHNOLOGYNETWORKS.COM Proteomics The meta-array of micropillars in the microfluidics device for enhanced immunoaffinity purification of HLA-restricted peptides. Credit: Xiaokang Li. Advancing Antigen Discovery With Microfluidics Automation for Sparse Samples Dr. Xiaokang Li and Prof. Dr. Michal Bassani-Sternberg, Ludwig Institute for Cancer Research, Lausanne Our group at the Ludwig Institute for Cancer Research in Lausanne, Switzerland, has published an innovative method in Cell Reports Methods for tumor antigen discovery. Our automated and cost-effective workflow in immunopeptidomics, utilizing microfluidics technology, overcomes limitations in sample preparation, particularly the immunoaffinity purification (IP) of human leukocyte antigen (HLA)-restricted peptides. This novel method enables sensitive detection of multiple immunogenic tumor-associated antigens from small clinical tumor biopsies, making it a powerful tool for antigen discovery in sparse samples. Reducing sample loss to increase immunopeptidomics sensitivity Mass spectrometry (MS)-based immunopeptidomics is a valuable method for identifying peptides presented by HLA molecules on cell surfaces, which are essential for T cell-mediated immune responses. The unique immunopeptidomes of cancer cells offer potential targets for immunotherapies like cancer vaccines and adoptive cell transfer therapies. MS is crucial for comprehensive peptide analysis, but current sample preparation methods for immunopeptidomics are laborious and hinder large-scale clinical applications. Existing alternatives, such as robotic platforms, are costly, posing financial burdens for many laboratories. The study aimed to develop an automated, cost-effective and efficient microfluidics-based workflow for sensitive immunopeptidomics to overcome these challenges. Automated sample prep with a microfluidics system We developed an automated and cost-effective workflow for immunopeptidomics by utilizing microfluidics technology to overcome the limitations of existing sample preparation methods, especially for the IP of HLA-restricted peptides. We engineered a microfluidic platform with a meta-array of micropillars to enhance immunoaffinity interactions. The platform incorporated a programmable fluidic control system for the IP procedure and integrated C18 cartridges for sample clean-up. This workflow streamlines the sample preparation process and reduces material consumption, leading to enhanced target purification. We demonstrated the performance of our approach by analyzing low-input samples and tumor biopsies, leveraging data-independent acquisition computational methods for sensitive detection of tumor antigens using MS. The novel microfluidics-based workflow showed competitive performance over the traditional one, offering an automated, cost-effective and efficient solution for immunopeptidomics analysis. 20 TECHNOLOGYNETWORKS.COM Proteomics The key findings of the paper were: • An automated microfluidics system with enhanced sensitivity for immunopeptidomics was created • Data-independent acquisition computational methods were able to provide in-depth and reliable immunopeptidomics analyses • A public spectral library constructed with published MS files enabled comprehensive analyses Reliable and sensitive peptide identification The newly created microfluidics-based workflow presents an automated and user-friendly system for immunopeptidomics. This is achieved by reducing the sample volume and integrating purification steps, resulting in enhanced assay efficiency. Additionally, with continuing developments allowing for scalability, larger-scale studies and potential clinical applications can be pursued in the future. Furthermore, in comparison to costly robotic liquid handling platforms, the microfluidics approach offers a cost-effective alternative, making immunopeptidomics analysis accessible to a broader spectrum of research laboratories. The combination of microfluidics technology and dataindependent acquisition computational approaches enables sensitive and reliable identification of tumor antigens. This can significantly contribute to the discovery of cancer-specific peptide antigens, which are crucial for developing targeted T cell-mediated cancer therapies, including TCR-T therapy and cancer vaccines. The ability to detect these antigens accurately and characterize them can potentially lead to the development of more effective personalized cancer treatments. Microfluidics devices’ ability to handle sub-milliliter sample volumes makes them suitable for analyzing limited clinical samples such as liquid biopsies or small tumor biopsies. This capability opens up opportunities for studying immunopeptidomes in situations where sample availability is limited. It can provide valuable insights into cancer-specific peptide antigens and immunogenic tumor-associated antigens, potentially leading to a better understanding of immune responses in cancer and the development of targeted therapies. The impact of this study lies in its advancements in the field of immunopeptidomics. By introducing an inexpensive, automated and easy-to-operate workflow using microfluidics technology, the study addresses bottlenecks in sample preparation and enhances target purification. This has significant implications for cancer research and personalized cancer therapies. The study enables more efficient and cost-effective analysis of tumor antigens, leading to the discovery of cancer-specific peptide antigens and immunogenic tumor-associated antigens. This knowledge can potentially revolutionize the development of targeted T cell-mediated cancer therapies, such as TCR-T therapy and cancer vaccines. Moreover, the ability to handle scarce clinical samples expands the possibilities for studying immunopeptidomes and improving our understanding of immune responses in cancer. While the study on the automated microfluidics workflow for immunopeptidomics brings significant advancements, it also has certain limitations that should be acknowledged. The evaluation of the workflow’s performance was mainly conducted using low-input samples and tumor biopsies. It is crucial to assess its applicability and performance across a broader range of sample types, including different cancer types and various biological fluids. Validation studies on a larger scale and diverse patient cohorts are necessary to establish the reliability and reproducibility of the approach. Another limitation is that the study does not address the potential challenges associated with analysis of peptides restricted by other HLA classes, such as Class II HLAs. Factors such as scalability, cost-effectiveness and integration with existing clinical workflows need to be considered for successful translation into large-scale clinical applications. Expanding the scope of application In the future, efforts should be directed towards scaling up the assay throughput by incorporating multiple microfluidics modules in a relatively cost-effective and small footprint. An intuitive system control interface and packaged chip devices should be implemented to allow inexperienced users to handle the platform effortlessly. To validate the clinical utility of the workflow, largescale validation studies involving a substantial number of patient samples should be conducted. This will enable the assessment of its diagnostic and prognostic capabilities and provide evidence for its effectiveness in personalized cancer treatment. Reference: Li X, et al. Cell Rep Methods. 2023;3(6):100479. 21 TECHNOLOGYNETWORKS.COM Credit: iStock Detecting Cancer From a Droplet of Blood Molly Campbell In 2000, the Human Genome Project (HGP) announced it had completed a working draft of the human genome sequence – a genetic “blueprint” of a human being. In the same year, a new project emerged, one that sought to look “beyond the genome”: The Human Protein Atlas, which aims to map all of the human proteins in cells, tissues and organs. A cell, tissue or organism’s proteome – the complete set of proteins it expresses at a given time – reflects its dynamic state more accurately than its DNA code. Genes could be turned “on” or “off”, proteins might undergo post-translational modification – processes that can determine, or perturb, a cell’s function. “Proteins are the building blocks of all life on this planet. They are also the targets for almost all drugs and thus incredibly important commercially,” says Mathias Uhlén, professor of microbiology at the Royal Institute of Technology (KTH) and program director of the HPA. “The holistic understanding of how proteins can execute all the functions in human life is still surprisingly unknown and thus a huge challenge for biomedical research.” Officially launched in 2003, the HPA integrates breakthrough technologies – including mass spectrometry (MS)-based proteomics, antibody-based imaging, transcriptomics and systems biology – to create an open access resource, which is used in over 150 countries. Uhlén adds: “The resource now consists of more than 5 million web pages and is updated annually with new data and features.” To date, the HPA has contributed to several thousands of publications across the life science fields and is recognized by ELIXIR as a “European core resource”. The structure of the HPA Over the last 20 years, the HPA has launched 10 different sections that each focus on varying aspects of human proteins: • Tissue – Showing the distribution of proteins across all major tissues and organs in the body • Brain – Exploring the distribution of proteins in various regions of the mammalian brain • Single-Cell Type – Studying expression of proteincoding genes in single human cell types using single-cell RNA sequencing (scRNA-seq) • Tissue Cell Type – Studying expression of proteincoding genes in human cell types based on bulk RNAseq data • Pathology – Showing the impact of protein levels on the survival of patients with cancer • Immune Cell – Showing expression of protein-coding genes in immune cell types • Blood Protein – Describing proteins detected in blood and secreted by human tissues Proteomics 22 TECHNOLOGYNETWORKS.COM Proteomics Credit: The Human Protein Atlas. • Subcellular – Showing the subcellular localization of proteins in single cells • Cell Line – Showing expression of protein-coding genes in human cell lines • Metabolic – Exploring expression of protein-coding genes in the context of the human metabolic network “The ‘flagship’ Tissue Atlas was launched in 2015 with more than 10 million microscope bioimages showing proteins on a single cell level across all major tissues and organs in the human body. The 2015 publication in Science now has ~10,000 citations and it is thus one of the most cited research publications in Europe during the last 10 years,” says Uhlén. In December 2022, version 22 of the HPA introduced two new sections that reflect the increasing utility of artificial intelligence (AI) and machine learning in proteomics research. Detecting cancer-associated proteins from a droplet of blood Version 22* of the HPA also introduced a new Human Disease Blood Atlas, which presents the results of a novel pan-cancer strategy to explore the proteome signature in blood samples obtained from cancer patients. For a disease caused by changes to the DNA code, it makes sense that genomics-based approaches have dominated cancer research over recent years. “However, to understand the consequences of these changes it is often necessary to instead study the protein levels in the tumor,” says Uhlén. This can be achieved using tumor biopsies, but the procedure is invasive and can cause distress for the patient. A blood-based test to explore protein expression is a desirable alternative. The researchers behind the Human Disease Blood Atlas analyzed 1,463 proteins from over 1,400 cancer patients. “We highlight proteins associated with each of the analyzed cancer types based on differential expression analysis as well as a machine-learning-based disease prediction strategy. By combining the results from all cancer types, a panel of proteins suitable for the identification of individual cancer types based on a drop of blood is presented,” Uhlén explains. To achieve this panel, a combination of proximity extension assay (PEA) technology and targeted proteomics (MS) was used. MS has long been considered the “gold standard” technology for high-throughput proteomics analysis in research settings. However, assays have been used in the clinical space for many years; examples of their application include the detection of troponin, which can indicate that a heart attack has occurred. “However, these assays are based on analysis of a single protein, but assays to detect and quantify multiple protein targets – with a sensitivity needed for the minute concentrations of proteins in the blood – have been lacking,” says Uhlén. Figure 1: Towards next-generation cancer prediction medicine. The example shows the elevated levels of the protein GFAP in the blood of patients with glioma. 23 TECHNOLOGYNETWORKS.COM Proteomics The introduction of next-generation blood protein profiling, based on parallel analyses of thousands of proteins at once, is changing the game. “PEA has allowed thousands of blood proteins to be analyzed from a small drop of blood. These developments mean that we are entering a new era for personalized medicine based on detailed multiplex blood protein profiling,” Uhlén adds. Such platforms are poised to have a major impact on the cost of diagnosing and treating cancer, a disease with a substantial economic burden. Next-generation blood proteome profiling – the future of clinical proteomics? Uhlén emphasizes that, while the first version of the Human Disease Atlas is focusing on cancer, the HPA will explore other diseases using the same approach in coming years, such as cardiovascular, infectious, neurodegenerative and autoimmune conditions. “This will make it possible to compare the blood profiles across major diseases and thousands of individuals,” he says. His words offer enthusiasm for a field that, despite significant advancements over the last decade, is yet to enter the clinical space on a widely adopted scale. “There are several obstacles to make these new findings translate into clinical practice. First, all potential tests based on new analytical platforms must be validated in independent cohorts and preferably with alternative analytical assays,” says Uhlén. This is no easy feat – it requires thousands of samples. The second key obstacle, according to Uhlén, is the regulatory requirements that are costly and timeconsuming. “It might be difficult to obtain clinical approval for panel tests measuring hundreds of proteins in parallel,” he says. “In addition, even very specific tests can give rise to ‘false positives’ causing unnecessary grief and psychological burden for the patient.” Therefore, clinically valuable tests must have secondary, independent tests available to identify “true positives” for diagnosis. These challenges aside, Uhlén is optimistic that with the rapid development of next-generation proteome profiling, “we will find ample applications in routine clinical practice in a few years.” As for the future of the HPA, it will continue to host its open-access resource to facilitate protein research around the world, adding data to complement the body-wide spatial profiling in cells, tissues and organs with data focusing on structure, interactions, modifications and function. Professor Mathias Uhlén was speaking to Molly Campbell, Senior Science Writer for Technology Networks. *Version 23 of the HPA was released in June of 2023.