Mathieu Lavallée-Adam, ECR Co-Chair, University of Ottawa, Canada

The HUPO Early Career Researcher (ECR) Initiative is proud to announce that Dr. Ruth Huttenhain and Dr. Mathieu Lavallée-Adam have been newly elected as its Co-Chairs. The ECR initiative also thanks Dr. Justyna Fert-Bober, founding member of the HUPO ECR Initiative and exiting Chair, for spearheading the activities that the ECR Initiative is now known for, such as the HUPO Mentoring Day, Manuscript Competition and Ph.D. Poster Competition. “I am happy to leave the leadership of the HUPO ECR Initiative in such good hands. I am convinced that Dr. Huttenhain and Dr. Lavallée-Adam will grow the initiative and build an environment that will foster new collaborations and bring new ideas to life,” declares Dr. Fert-Bober. Dr. Huttenhain, who has, among other things, organized the HUPO ECR manuscript competition in the past, says: “In my role as a Co-Chair of the HUPO ECR Initiative, I am particularly excited about growing a larger community of early career scientists in proteomics with the ultimate goal to provide guidance for taking the next steps in their careers. I will also be honored to represent the ECR Initiative in the Organizing Committee of the HUPO World Congress and to advocate for a program that provides visibility for early career scientists and highlights their scientific contributions.” Finally, Dr. Lavallée-Adam, who has been organizing last year’s mentoring sessions at HUPO CONNECT and coordinating ECR’s HUPOST publications adds: “Early career scientists are more diverse than ever and the HUPO ECR Initiative must help them exploit their full potential. I look forward to building upon our current portfolio of activities and constructing new initiatives to showcase proteomics rising stars and provide them the tools to be more competitive on the job market.”

You will find below short bios of Dr. Huttenhain and Dr. Lavallée-Adam, who are both recipients of the Proteomics Highlight of the Year by an Early Career Researcher Award given at the 2018 Human Proteome Organization World Congress.

Ruth Huttenhain
Ruth Huttenhain is an Assistant Professor at the University of California, San Francisco (UCSF) in the Department of Cellular and Molecular Pharmacology. She obtained a Pharmacy degree from the University of Bonn and a PhD from ETH Zurich, Switzerland, where she developed high-throughput, large-scale targeted mass spectrometric approaches. During her postdoc at UCSF, Ruth extended her expertise in quantitative mass spectrometry to study dynamics of protein interaction networks. She pioneered a proximity labeling-mass spectrometry approach that simultaneously captures the precise temporal remodeling and spatial organization of proximal protein networks. The research of Ruth’s research group at UCSF focuses on characterizing protein interaction and signaling networks to understand the biology underlying the development of psychiatric disorders and the sensing and transmission of pain.

Mathieu Lavallée-Adam
Mathieu Lavallée-Adam is an Assistant Professor at the University of Ottawa in the Department of Biochemistry, Microbiology and Immunology and is affiliated to the Ottawa Institute of Systems Biology. He obtained a B.Sc. in Computer Science and a Ph.D. in Computer Science, Bioinformatics option, from McGill University. He then performed his postdoctoral research at The Scripps Research Institute. His research focuses on the development of novel statistical and machine learning algorithms for the analysis of mass spectrometry-based proteomics data and protein-protein interaction networks. He also designs computational methods mining proteomics datasets for biological information through their integration with genomics data. Dr. Lavallée-Adam is a recipient of the John Charles Polanyi Prize in Chemistry, recognizing the impact of his bioinformatics algorithms on the mass spectrometry community. He is also a member of the Board of Directors of the Canadian National Proteomics Network (CNPN), in which he mentors a group of early career researchers in the establishment of activities for the Canadian Proteomics community.

Mathieu Lavallée-Adam, University of Ottawa, Canada

Blandine Chazarin is a postdoctoral scientist in Dr. Jennifer Van Eyk’s laboratory at Cedars-Sinai hospital in Los Angeles, California. After four years in Paul Sabatier University in Toulouse studying Biology, Blandine explored proteomics for a year before beginning a Ph.D., in which she used mass spectrometry to better understand hibernating brown bear physiology, with the goal to discover new therapeutic approaches for muscle atrophy. In October 2019, she obtained her Ph.D. in LSMBO lab in Strasbourg, France. Blandine has also been President of the Youth Club of the Proteomic French Society for three years and was the organizer of yearly meetings dedicated to young scientists in proteomics. She is now involved in the Postdoctoral Scientist Society at Cedars-Sinai and continues to use mass spectrometry to answer biological questions.

Professor Vera Ignjatovic, Chair, HUPO Marketing and Membership Committee

Vera is a Senior Principal Research Fellow and a group leader of the Haematology team at the Murdoch Children’s Research Institute in Melbourne, Australia. She lead a highly productive, internationally competitive research team in the field of paediatric thrombosis and haemostasis (aka kids' blood). Her research efforts include the use of proteomic studies in the paediatric setting, where she established for the first time the concept of Developmental Proteomics.

As a lifelong learner she recently completed a Global Executive MBA at the Monash University in Melbourne, an experience that included training in strategic marketing; executive leadership; corporate finance, governance and strategy; as well as innovation and entrepreneurship, and commercialisation of technology. The learnings from the Global Executive MBA and exposure to the world outside of the immediate research environment will be of advantage in her role as the incoming Chair of the HUPO Marketing and Management Committee.

Whilst the 2021 Marketing and Membership committee is in its infancy, Vera is very happy to announce 5 new committee members, James Waddington (Co-Chair), Conor McCafferty (PhD representative), Jennifer Geddes-McAlister, Benjamin Garcia and Lennart Martens. She very much looks forward to working with all HUPO Marketing and Membership Committee members to increase the visibility of the HUPO brand in 2021 and beyond.

Mathieu Lavallée-Adam, University of Ottawa, Canada

Emily Hashimoto-Roth is a graduate student at the University of Ottawa pursuing a Master’s in Biochemistry specializing in Bioinformatics, under the co-supervision of Dr. Mathieu Lavallée-Adam and Dr. Steffany Bennett. Having obtained an H.B.Sc. in Biopharmaceutical Science at the University of Ottawa, her current research focuses on the application of statistical models and machine learning algorithms to mine and analyze protein-protein interaction datasets. She is a trainee within the NSERC-CREATE Metabolomics Advanced Training and International Exchange Program (MATRIX), further diversifying her graduate studies. In addition to her studies, she is the Director of Communications for a Canadian federal non-for-profit organization called Pulsar Collective, whose mission is to improve gender equality in STEM. She is also a member of the Canadian National Proteomics Network, which works to advance the proteomics field by fostering a community for researchers to meet and collaborate.

Once again this year, the HUPO Early Career Researcher (ECR) initiative in collaboration with the EuPA Young Proteomics Investigators Club (YPIC), organized mentoring activities during HUPO Connect 2020, where early career researchers could hear from and interact with leaders of the Proteomics field. This year, the mentoring sessions were integrated in the main program of the congress at different time slots to allow people across to globe to connect and exchange. In the first session, trainees and supervisors learned how to get the best out of a mentor-mentee relationship with Lisa Jones (University of Maryland) and Brandon T. Ruotolo (University of Michigan). In the second session, Benjamin Garcia (University of Pennsylvania), Ruth Huettenhain (University of California San Francisco), and Justyna Fert-Bober (Cedars-Sinai) discussed work-life balance and the challenges faced during the COVID-19 pandemic. Finally, in the last session, Bernard Delanghe (Thermo Fisher Scientific) and James Anson (Molecular Omics) gave insights about the transition from academia to the industry.

With over 200 attendees participating in all three sessions, the 2020 mentoring activities show the greatest attendance since the beginning of mentoring activities at the HUPO conferences. We thank all mentors for sharing their experience with mentees. We also thank all attendees for their participation and for engaging in stimulating, thought provoking discussions during the congress.

The HUPO Early Career Researcher (ECR) initiative and the EuPA Young Proteomics Investigators Club (YPIC) held a virtual poster session at HUPO Connect 2020 for the finalists of the 2020 Ph.D. poster competition sponsored by Molecular Omics. Eight abstracts were selected by an international panel to give their authors an opportunity to present their work in 5-minute oral presentations during a dedicated Ph.D. Poster Competition session at HUPO Connect 2020. The eight finalists were: Edwin Escobar, University of Texas at Austin, Austin, United States; Xiaobo Tian, University of Groningen, Groningen, Netherlands; Joshua Charkow, University of Toronto, Toronto, Canada; Ugo Dionne, Centre de recherche du Centre Hospitalier Universitaire (CHU) de Québec-Université Laval, Quebec, Canada; Andikan Nwosu, Brigham Young University, Provo, United States; Maria Jassinskaja, Division of Molecular Hematology, Lund Stem Cell Center, Lund University, Lund, Sweden; Ana Montero Calle, Functional Proteomics Unit, UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, Madrid, Spain and Jessica Nickerson, Dalhousie University, Halifax, Canada. All finalists gave very high-quality presentations, which made for an extremely interesting session. In the end, Ugo Dionne took 1st place ($300), Maria Jassinskaja finished 2nd ($150), and Edwin Escobar was recognized with the 3rd place ($150). Congratulations to all finalists! You can read more about the finalists here.

The HUPO Early Career Researcher (ECR) initiative and the EuPA Young Proteomics Investigators Club (YPIC) joined forces for HUPO Connect 2020 to organize the sixth edition of the ECR manuscript competition to select the Proteomics Highlight of the Year by an early career researcher. Every year, early career proteomics researchers are invited to participate to this competition by submitting a manuscript they published in the previous year. An external committee then selects three finalists, who present their work at the HUPO World Congress. Their talks are then evaluated to determine the winner of the competition.

This year's finalists were Marian Kaloscay (Harvard Medical School) Maria Robles (Ludwig Maximilian University of Munich), and Jakob Trendel (Technical University Munich). The dedicated oral presentation session for the manuscript competition was one of the highlights of HUPO Connect 2020 with three high-quality talks followed by dynamic Q&A sessions. The quality of the presentations made it very difficult for the panel of external judges to select the overall winner of the competition. Ultimately, this year’s Proteomics Highlight of the Year by an early career researcher was awarded to Maria Robles for her work entitled: “Sleep-wake cycles drive daily dynamics of synaptic phosphorylation”. Congratulations to all! You can read more about the finalists here.

By Michelle Hill, QIMR Berghofer Medical Research Institute, Brisbane, Australia, and Stephen Pennington, University College Dublin, Dublin, Ireland.

Developing new therapies for clinical use is a long and costly process. Optimizing dose and schedule in early clinical trials and selecting the right patients for the therapy are two critical aspects for the drug development process. Read how Dr Henrik Neubert (Pfizer), Dr Amanda Paulovich (Fred Hutchinson Cancer Research Center) and Dr Carl Barrett (Astra Zeneca) have used targeted proteomics to facilitate drug development.

In the words of Dr Carl Barrett (Astra Zeneca), “90% of drugs fail for 2 reasons, bad chemistry or bad biology”. Protein technologies have been central in drug development, as most drug targets and their downstream effectors are proteins. In recent years proteins (often antibodies) are also being used as new therapeutics. Recently, proteomics technologies have matured to the stage where they are now sufficiently robust and reproducibly that they being developed into robust high throughput assays. Proteomics can be key to helping to identify bad chemistry (of the drug), and to illuminate bad biology by facilitating pharmacodynamic (PD) and proof of mechanism (POM) studies.

Selecting therapeutic targets with the right properties

With PhD and postdoctoral training in quantitative protein mass spectrometry, peptide synthesis and MALDI surface chemistry, it was natural for Dr Hendrik Neubert to bring his proteomics knowledge to drug development when he joined Pfizer in 2004. Hendrik now leads a translational biomeasures and protein biomarkers group that develops proteomics assays based on mass spectrometry to measure synthesis rates and concentrations of therapeutic targets as well as their engagement with biotherapeutics. This proteomic data when combined with critical experimental data enables mechanistic modeling to support key drug development program decisions such as the feasibility of modulating the activity of a particular protein target or establishing drug dosing regimens. Hendrik and his team have been particularly influential in the area of immunoaffinity mass spectrometry and worked to robustly position this technology in early clinical trials of Pfizer’s drug candidates. His team was the “terminator” for Pfizer’s osteopontin neutralizing antibody program due to undesirable target properties.

Osteopontin seemed to be a perfect drug target and it was being pursued by several biopharmaceutical companies. It is a circulating protein and pre-clinical studies implicated it in many immune-related diseases, liver fibrosis and cancer. In a cynomolgus monkey model, a neutralizing antibody to osteopontin was effective in ameliorating arthritis. The literature describes a successful phase I trial demonstrating safety and tolerability in men, however, no improvement in disease was observed in a subsequent Phase IIA trial on rheumatoid arthritis.

The Pfizer team was interested in developing a neutralizing antibody against osteopontin for a different indication but before proceeding, Neubert’s team was tasked to investigate the potential PK/PD risks associated with the osteopontin target. The team realized that the half-life of osteopontin in human blood had never been determined before. This is an important parameter for a neutralizing antibody, because if osteopontin protein is quickly eliminated from the body, then the amount and frequency of antibody dosing required for efficacy may not be feasible therapeutically.

Protein half-lives were previously measured by injecting radioactively or fluorescently labelled recombinant protein into preclinical models. Unfortunately, this method is really inadequate for a reliable physiologically relevant half-life measurement as it only measures the rate of elimination, the protein being measured is not endogenous and is typically administered above endogenous concentrations. Most importantly the approach cannot be applied to humans!

With their proteomics expertise, Neubert’s team used longitudinal serum samples from a previously conducted stable-isotope labelled leucine (13C-Leu) pulse-chase study in humans and enriched osteopontin using an anti-osteopontin antibody prior to tryptic digestion. Liquid chromatography-tandem mass spectrometry of a proteotypic peptide with and without 13C-Leu incorporation was used to determine the half-life of osteopontin in human blood from healthy individuals. Surprisingly, with a half-life of around just 20 minutes osteopontin has one of the shortest half-lives Neubert’s team has ever observed for a human protein. Considering the rapid synthesis and clearance from human blood it was evident that the dosing schedule for a neutralizing antibody that would be required was simply not feasible. Hence, Pfizer stopped the discovery project very early before significant further investments were made. Of course, pharma teams all want their therapeutic programs to be successful, but if a drug development program does fail it is ideal it fails early to avoid costly clinical studies so resources can be spent on programs that are more likely to succeed.

Elucidating tumor biology and drug mechanisms of action

Quantifying proteins and protein networks for pharmacodynamic and proof of mechanism studies is critical for translating novel therapies, to confirm the biological mechanisms underlying new compounds and to inform drug dose and scheduling in clinical trials. Immunohistochemistry (IHC) is generally the preferred technology for these studies because it is sensitive, provides biomarker spatial distribution and is semi-quantitative. For clinical implementation, IHC is a well-understood analytical modality. However, IHC is critically dependent on the absolute specificity of individual antibodies, and establishing this specificity is costly in terms of time and resource. As a result, only a handful of fully validated IHC protocols can be developed for each drug project, where the choice of which IHC assays to develop is largely done on the basis of “best educated guess” arising from orthogonal preclinical methods such as Western blotting.

Adding to the challenge of measuring specific proteins by IHC, it is recognized that proteins act as interconnected networks, and the effects of cancer driver mutations for example spread throughout the networks. Ideally we would have assays to quantify panels of multiple proteins in early phase clinical trials to assess the activity of pathways/networks that determine treatment responses, and this developmental effort is not practical using IHC

More specific and quantitative “NextGen” proteomic techniques are now starting to be used, exemplified by the stimulating interdisciplinary collaboration between Dr. Carl Barrett (AstraZeneca) and Dr. Amanda Paulovich (Fred Hutchinson Cancer Research Center). Paulovich is a geneticist and oncologist who has run a translational proteomic laboratory at Fred Hutch for the past 17 years. Barrett has a PhD in biophysical chemistry and is VP Translational Sciences Onc iMed at AstraZeneca.

A recent collaborative project between their teams identified phospho-RAD50 as a novel pharmacodynamic biomarker for inhibitors of DNA damage checkpoint signaling kinases ATM and ATR, which are being tested in clinical trials in a variety of cancers. While pharmacodynamic biomarkers were available, the assays and biomarkers were not ideal.

Paulovich’s lab developed a multiplexed immuno-multiple reaction monitoring mass spectrometry assay to measure proteins and phosphoproteins in the signaling cascade downstream of the DNA damage checkpoint. The team used this targeted mass spectrometry-based assay panel to identify Ser635-phosphorylated RAD50 as a novel pharmacodynamic biomarker of ATR and ATM kinase inhibitor pharmacology. The pRad50 biomarker was further validated by Barret’s team using two preclinical xenograft models and using archived human tumor material. Together this supported clinical utilization of pRAD50 as a biomarker to probe clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.

Towards broader clinical testing

The productive interdisciplinary academia-pharma collaboration between Barrett and Paulovich has propelled immuno-MRM assays from research to established Clinical Laboratory Improvement Amendments (CLIA)/clinical grade assays, suitable for clinical trials to facilitate drug development. Paulovich’s team has developed >1,400 targeted mass spectrometry-based assays, which her laboratory runs in its recently-established CLIA environment. CLIA establishes the quality laboratory framework for human diagnostic testing, so Paulovich is taking targeted proteomics assays a step closer to clinical use.

Geoffrey G. Hesketh¹ & Anne-Claude Gingras^1,2
1Lunenfeld-Tanenbaum Research Institute, Sinai Health System
²Department of Molecular Genetics, University of Toronto
Toronto, Ontario, Canada

Cells can be viewed as functioning in four dimensions: three-dimensional space (structural organization) and one-dimensional time (dynamics). To understand cell function, we must therefore understand how their constituent macromolecules, including proteins, lipids, carbohydrates and nucleic acids, exist in space as well as in time. Here, we discuss the use of proteomics methods to probe such questions.

The era of molecular cell biology (i.e., studying the mechanisms by which molecules orchestrate cell function) was ushered in during the 1950s with the development of two key techniques – cellular electron microscopy and cell fractionation by differential centrifugation. By combining these powerful approaches with existing classical biochemistry methods, critical insight into how cells are organized into distinct membrane-bound organelles (e.g., ER, Golgi, mitochondria, lysosomes) and molecular structures and machines (e.g., chromatin, ribosomes) was gained. Importantly, this led to the realization that these different compartments carry out distinct cellular functions, largely due to the differential partitioning of specific macromolecules to distinct compartments.

In the late 1990s and early 2000s, genome sequencing and accelerated development in mass spectrometry technologies for peptide sequencing set the stage for the new field of proteomics, which was ideally suited to addressing molecular cell biology questions. Rather than analyzing differential cell fractions by low throughput biochemistry methods, the ability to assign proteins en masse to distinct fractions (and therefore distinct cell compartments) became possible. The speed, sensitivity, and resolution of mass spectrometers dramatically improved over the years, and when combined with modern proteomics technologies (including multiplexed quantitation by isotope tagging strategies and improvements in data analysis), spatial proteomics was able to develop into a mature field1.

An inherent limitation to spatial proteomics methods that rely on cell lysis and differential fractionation is that spatial information is captured after both lysis and fractionation have occurred. Lysis necessarily involves some form of membrane disruption (often by mechanical means and the use of detergents) and the fractionation (the stage at which ‘spatial’ information is captured) is carried out under non-native conditions. While the integrity of certain cellular compartments may be maintained during such procedures, this is not universally true for all compartments. Furthermore, it is becoming increasingly appreciated that many cell functions are driven by extensive contacts between distinct organelles2, and these associations are poorly captured by lysis and fractionation approaches.

Methods that bypass the requirement for isolating intact structures prior to identification by mass spectrometry can overcome many of these limitations. The first practical application of proximity-dependent biotinylation followed by mass spectrometry (BioID) was described in 20123; reviewed in4). BioID employs a bacterial biotin ligase (BirA) in which a single point mutation (R118G) yields an abortive enzyme (BirA*) that creates a ‘cloud’ of reactive biotinyl-AMP. This allows biotinylation of proteins on accessible lysine residues in the vicinity of the BirA*-tagged bait (estimated to be within a ~5 nM radius in one study5). Since the introduction of BioID, the proximity-dependent biotinylation enzyme toolbox has grown to include peroxidase-based enzymes (e.g., APEX2) that can label tyrosines6, biotin ligases from other species7,8 and more catalytically efficient versions of the BirA enzymes generated through directed evolution (such as TurboID and miniTurbo)9. A key feature of proximity-dependent biotinylation approaches is that spatial information is captured in living cells prior to lysis, through covalent labeling of the proximal proteins. In this case, organelles and protein interactions do not need to be maintained during lysis and purification, and as such a view of the ‘neighborhood’ of a protein of interest is obtained inside living cells.

By definition, proximity-dependent biotinylation provides an assessment of the distance relationship between a bait and a prey (three-dimensional space). Importantly, labelling can be carried out in a specific window of time, and the use of enzymes with labelling kinetics on the order of minutes (e.g., APEX2, miniTurbo and TurboID) allows for the design of experiments with a temporal perspective. With careful experimental design, spatio-temporal relationships may therefore be decoded through a variety of data analysis approaches.

We and others have begun employing proximity-dependent biotinylation approaches to map organelles and other structures in space and time. When analyzed with tools for cell localization ontologies (such as GO cellular component), proximal preys help reveal the localization of the bait to specific compartments. However, the quantitative recovery of individual preys by two baits with superficial localization to the same structure is not necessarily identical, but rather reflects the respective organization of the baits and preys within the structure10. Preys that directly bind to a bait, or are in close proximity to it within a protein complex, tend to be more strongly labeled than more distant preys within the same structure. While there is rarely sufficient information in a single bait BioID experiments to untangle these complex relationships (also see4,11 for discussions), analyzing large datasets in aggregate has enabled reconstruction of the organization of several structures and organelles – including the centrosome-cilium12, stress granules and P-bodies10, and more recently the mitochondria13. By expanding the analysis to include baits localizing across multiple distinct compartments throughout the cell we have begun to create a global ‘proximity-map’ of a cell, localizing over 4000 proteins to distinct cellular locations14 (see humancellmap.org). Expansion of these studies to explore dynamic changes in subcellular organization will constitute a stimulating challenge for experimental design, execution and data analysis.

In recent work, our group has also explored the use of BioID baits as ‘organelle sensors’ to evaluate changes in organelle proteomes following pharmacological or genetic perturbations. BioID labelling profiles are highly reproducible across experiments, and therefore performing BioID experiments with a given ‘sensor’ under different conditions (e.g., knocking-out a gene of interest, drug treatment, differential cell growth conditions) can illuminate dynamic changes that occur on specific organelle surfaces. Leveraging this approach, we used the lysosomal R-SNARE proteins VAMP7 and VAMP8 as ‘sensors’ to identify proteins localizing to the cytosolic face of late endocytic membranes (i.e., late endosomes, lysosomes, and the product of their fusion, endolysosomes) (see Figure 1). This strategy revealed novel relationships between lysosome membrane trafficking complexes and proteins involved in nutrient signalling through the large kinase complex mTORC1 (mechanistic Target Of Rapamycin, complex 1). Our follow-up studies demonstrated an unanticipated interplay between two key mTORC1 activation pathways – namely, activation by exogenous amino acids and by lysosome-derived amino acids. Importantly, the latter pathway is implicated in the growth of Ras-driven cancer cells, which can use lysosome-derived amino acids (acquired through macropinocytosis of exogenous protein) to fuel their growth. It is likely that the concept of BioID ‘organelle sensors’ will find wider application in cell biology over the coming years.

In summary, proximity-dependent biotinylation approaches offer complementary views to fractionation approaches in spatio-temporal proteomic studies. Their expanded use will allow increasingly complex cell biological questions to be answered directly in living cells.

Legend

Figure 1 – The use of BioID ‘organelle sensors’ to map the surface proteomes of late endocytic organelles.

Bio

Dr. Geoffrey Hesketh is a postdoctoral fellow in Dr. Anne-Claude Gingras’ lab at the Lunenfeld-Tanenbaum Research Institute in Toronto, where he uses proteomic methods to explore how lysosomes control cell growth. He was previously a postdoctoral fellow in Dr. Paul Luzio’s lab at the Cambridge Institute for Medical Research at the University of Cambridge in the UK, where he developed his interest in lysosome biology by studying mechanisms of late endosome-lysosome fusion and recycling. Prior to this, he obtained his PhD in Dr. Jennifer Van Eyk’s lab at The Johns Hopkins University School of Medicine.

References

1. Lundberg, E. & Borner, G. H. H. Spatial proteomics: a powerful discovery tool for cell biology. Nat. Rev. Mol. Cell Biol. 1 (2019). doi:10.1038/s41580-018-0094-y

2. Prinz, W. A., Toulmay, A. & Balla, T. The functional universe of membrane contact sites. Nat. Rev. Mol. Cell Biol. 21, 7–24 (2020).

3. Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–10 (2012).

4. Samavarchi-Tehrani, P., Samson, R. & Gingras, A.-C. Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches. Mol. Cell. Proteomics 19, 757–773 (2020).

5. Kim, D. I. et al. Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc. Natl. Acad. Sci. U. S. A. 111, E2453-61 (2014).

6. Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

7. Kim, D. I. et al. An improved smaller biotin ligase for BioID proximity labeling. Mol. Biol. Cell 27, 1188–96 (2016).

8. Ramanathan, M. et al. RNA-protein interaction detection in living cells. Nat. Methods 15, 207–212 (2018).

9. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

10. Youn, J.-Y. et al. High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies. Mol. Cell 517–532 (2018). doi:10.1016/j.molcel.2017.12.020

11. Gingras, A.-C., Abe, K. T. & Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr. Opin. Chem. Biol. 48, 44–54 (2019).

12. Gupta, G. D. et al. A Dynamic Protein Interaction Landscape of the Human Centrosome-Cilium Interface. Cell 163, 1483–1499 (2015).

13. Antonicka, H. et al. A High-Density Human Mitochondrial Proximity Interaction Network. Cell Metab. 32, 479-497.e9 (2020).

14. Go, C. et al. A proximity biotinylation map of a human cell. (2019). doi:10.1101/796391

By Svetlana Murzina, Karelian Research Centre of the Russian Academy of Sciences, Petrozavodsk, Russia, and Michelle Hill, QIMR Berghofer Medical Research Institute, Brisbane, Australia

Take a journey with us across three Russian lakes and learn how Dr Polina Drozdova, Dr Ekaterina Borvinskaya and PhD student Albina Kochneva are using proteomics to understand the wonderful aquatic ecosystem. Their research starts with field-trips for sample collection, from colorful crustaceans to the not-so-colorful fish tapeworms.

Proteomics research is increasing the understanding of fundamental issues in ecology and parasitology, and also has important applications in pharmacology and agriculture, to identify new targets for antihelmintic drugs including those against tapeworms of fish.

Location 1: Irkutsk, Baikal Lake, Siberia

Lake Baikal is a fascinating place for anyone, and especially for those interested in molecular ecology. The deepest and oldest lake on Earth is home to 300+ species of small crustaceans (gammarid amphipods). They occupy various niches, differ in size and diet, and, most interestingly in color, which varies from transparent and milky white to bright orange, red, blue, and even dark violet.

These bright colors triggered Dr Polina Drozdova to move across half the country to pursue Baikal research - after her first summer trip to Baikal in 2012.

Polina recently obtained support from the Russian Science Foundation to study coloration and vision of endemic Baikal amphipods.

“As a starting point, we chose a very abundant species Eulimnogammarus cyaneus that looks like a result of a natural experiment. The majority of individuals have blue bodies, exactly as the species epithet suggests, but sometimes orange individuals can be found. Importantly, if protein integrity is in any way compromised in samples of the blue animals, the color changes into orange. This change reminds the mechanism well-known for crayfish and lobsters, which turn bright red when cooked due to the degradation of carotenoid-binding proteins called crustacyanins. This similarity motivated us to search for possible proteomic differences between animals of different colors. Indeed, we found two proteins, levels of which were much higher in blue animals that in the orange ones” said Polina. 

The proteomics results were astounding. Instead of crustacyanins, Drozdova’s team found the proteins share similar domains to insect pheromone/odorant-binding proteins that recognize a wide range of hydrophobic molecules.

“Even though carotenoids have not been included in this range, it was logical to suggest that the amphipod proteins (let us call them crustacyanin analogs) bind carotenoids. Indeed, further experiments supported this hypothesis” – said Polina.

These fascinating results were recent published, and Polina’s team plan to dig deeper into the molecular mechanism underlying carotenoid binding by these proteins and explore the diversity of these proteins in species with different body colors.

Location 2: Petrozavodsk, Onego Lake, Karelia

More than 4,000 km away, two young biochemists at the laboratory of environmental biochemistry IB KarRC RAS in Karelia are using proteomics to tackle a practical problem important for aquaculture and fishery sciences.

Dr Ekaterina Borvinskaya explains “Helminths of the order Bothriocephalidea are parasites of marine and freshwater teleost fish common throughout the world.”

“Like many other researchers, we work with non-model organisms for which there is no transcriptome, genomic, or proteomic data. Thus, we first decided to assemble the de novo transcriptome and annotate it for the tapeworm T. nodulosus, a common parasite of Holarctic freshwater fish. In our recent publication in Marine Genomics, we presented a functional annotation of transcripts and predicted the parasite proteome. Amazingly, in cestodes, about two-thirds of proteins is known to differ significantly in structure and, therefore, functions from proteins of other living organisms. Analysis of the T. nodulosus transcriptome revealed about a quarter of proteins with a completely unique structure even in comparison with other studied flatworm species. Such incredible biochemical diversity represents a huge parasite taxon that is very hard to get used to! However, the findings gave us an idea of a separate universe of cestodes with various unknown biologically active compounds in it”- said Ekaterina.

PhD student Albina Kochneva first joined the laboratory for her Bachelor Thesis, and has been researching parasitic worms of the genus Triaenophorus ever since. After successfully completing her Masters thesis using proteomics to study T. nodulosus and antioxidant protection enzymes of its intermediate host perch (Perca fluviatilis), Albina is now pursuing PhD in parasite proteomics.

“I became interested and did not stop wonder to how these amazing organisms adapt to life inside another organism.” - said Albina.

She studied two species of the Cestoda class which live in the same ecosystems and may infect the same definitive host (the same one fish), Triaenophorus nodulosus and Triaenophorus crassus. But, through the evolution, these tapeworms were “spatially” dispersed: T. nodulosus larvae are able to infect in the liver parenchyma of a very wide range of second intermediate hosts (different fish species) while T. crassus larvae almost exclusively inhabit the muscles of fish of Salmonidae family.

To understand the mechanisms of adaption, Albina compared the protein profiles of T. nodulosus and T. crassus at different life stages, and in different segments of the parasite body.

“To confirm the biochemical heterogeneity of different parts of the worm's body, we applied 2D-DIGE electrophoresis and LC/ESI-MS/MS together with analysts from St. Petersburg State University (St. Petersburg, Russia). We found that there is a quantitative and qualitative variability of some proteins in different parts of the parasite's body, which distinguishes and maintains their morphological and physiological characteristics.” – said Albina.

These results were presented at the 11th International Conference of Bioinformatics of Genome Regulation and Structure \ Systems Biology in 2018.

Besides these finding, it was revealed the huge amount of the secreted protein with unknown function in the head of the plerocercoid larva of T. nodulosus. This protein was completely absent in T. crassus. It was assumed that the protein might be responsible for the attachment and co-exist of T. nodulosus with its various host via the liver parenchyma. In contrast, T. crassus is unable to locate in the liver and performs specialization to its host.

Furthermore, proteomics analyses of larval stage T. nodulosus collected from the liver of different species of fish (perch Perca fluviatilis L., ruffe Gymnocephalus cernuus L. and burbot Lota lota L) revealed that the expression of some proteins at the same development stage depends on the environment (host-specific). These results support the Red Queen Hypothesis by Valen (1973) on the co-evolution of parasites and their hosts.

“For several hundred million years, the threat of infection by cestodes (tapeworms) has been a factor in the evolution of vertebrates and, definitely, to some extent, affect the formation of this taxon. This never-ending “attack and defense” processes are realized with contrivances at the molecular level resulting in inevitable reactions and inventions on both sides” – said Ekaterina

Location 3: Freshwater lakes, White Sea Basin, Kola Peninsula

The team recently turned their attention to the highly complex lifecycle of the helminth Schistocephalus solidus (Cestoda), which journeys from host to host via the trophic net, inhabiting two categories of environment: the first order is “inside the host” or internal environment, and the second order – “the host external environment”.

“The parasite transfers from its intermediate hosts - poikilothermic animals: zooplankton, a representatives of cyclopoid copepods, to fish, the three-spined stickleback (Gasterosteus aculeatus), and finally to the homeothermic animals, usually fish-eating birds.” – explains Ekaterina.

“Indeed, for the first time, the temperature-induced re-organization of proteins and lipids of S. solidus during the transition from the fish host to warm-blooded host will be carried out. The studies have already been done at the transcriptome level but we are interested in direct registration of biomolecules of the parasite and its host, especially at the surface of the parasite body, which are regions of active metabolic exchange or communication with the host. It should be noted, that such biological task can be satisfied only by analysis of proteome and lipidome together. These molecules maintain the interactions of organisms with the environment whether in a parasitic, symbiotic, or trophic activity. The proteomic analysis will be performed with the participation of specialists from the “Human Proteome” Core Facility of the Institute of Biomedical Chemistry (IBMC, Moscow, Russia)” - said Ekaterina.

But, the research starts not in the laboratory but in the field. For this, Albina was fishing the three-spined stickleback in the freshwater lakes of the White Sea Basin in June. She needs to develop good fishing skills because three-spined stickleback adults swim fast and are hard to catch! Albina also maintains an aquaria for three-spined stickleback in the laboratory.

Apart from the essential outdoor activities, conferences and practical schools offered by HUPO, RHUPO and EuPA have also been essential aspects of Albina’s scientific life. “All these activities help me to meet friends and colleagues in the field, to tell about my results and getting valuable feedback, to follow the announcements about the recent achievements in mass-spectrometry and proteomic approaches. It is interesting to think how to apply it for my research” - tells Albina.