This year, the XI Proteomics Workshop will present lectures from specialists from six countries.
Amanda Paulovich, Fred Hutch, Seattle, USA
Title: Using Proteomics to Build NextGen Diagnostic Solutions for Precision Oncology
Summary: While the current genome-centric approach to Precision Oncology has extended the lives of subsets of patients, many patients do not respond to the selected therapy, and many whose tumors initially respond have a high chance of recurrence as resistant disease. New approaches are required to capture complex clinical phenotypes and better match patients to efficacious therapies. Novel strategies will be discussed to identify NextGen diagnostics incorporating mass spectrometry-based protein assays.
André Zelanis, UNIFESP, Sao Paulo, Brazil
Title: Prospection of markers associated with proteolytic processing in biological samples of patients with melanoma
Summary: Melanoma is aggressive skin cancer and a lethal melanocytic neoplasm with an increasing annual number of cases. Since the protein composition (proteome) of tumoral cells may be regarded as a description of the cellular status, the evaluation of protein expression patterns in biopsy samples may lead to the discovery of cancer-related markers and new drug targets. In the context of tumoral development, proteolytic processing plays an important role in the dispersion of tumor cells to distant sites (metastasis) as well as in mediating irreversible signaling events. Therefore, we interrogated the Biobank of the São Paulo of Cancer Institute (ICESP) aiming at the prospection of molecular markers derived from proteolytic processing events. We applied an N-terminomics approach to investigate proteolytic processing events in plasma samples of patients with early/localized or systemic melanoma. Plasma samples from melanoma patients were subjected to gelatin-zymography and significant activity was found in samples from systemic disease, indicating active gelatinases. Albumin and IgG-depleted plasma samples were submitted to Terminal Amine Isotopic Labeling of Substrates (TAILS) and the corresponding prime-side peptides were analyzed by LC-MS/MS and database searching. We identified > 100 unique peptide signatures, corresponding to proteolytically processed substrates in each disease state (early/localized or systemic melanoma): the majority of them were derived from ordinary plasma proteins, such as fibrinogen, complement proteins, and apolipoproteins among others. Prime and non-prime subsite specificity mapping of the peptide set suggested the activity of arginyl-hydrolases. In conclusion, data obtained in this work provided a systems-wide profile of proteolytic signalling in melanoma, with important translational potential for disease prognosis.
Anthony Purcell, University of Monash, Melbourne, Australia
Claire Eyers, The University of Liverpool, Liverpool, United Kingdom
Title: Phosphorylation-mediated signalling – a new era?
Claudio Iacobucci, Martin Luther University Halle-Wittenberg, Germany
Title: In-Vitro and In-Cell Cross-Linking/Mass Spectrometry: from 3D-Protein Structure Investigations to Proteome-Wide Interactome Studies
Summary: Mass spectrometry (MS) has become one of the key technologies of structural biology. In this presentation, the contributions of chemical cross-linking combined with mass spectrometry (XL-MS) for studying three-dimensional structures of proteins and for investigating protein-protein interactions are outlined. We summarize the most important cross-linking reagents, software tools, and XL-MS workflows and highlight prominent examples for characterizing proteins, their assemblies, and interaction networks in vitro and in vivo. The special focus is on MS-cleavable cross-linkers and integrated workflows we have developed over the years to perform XLMS at different levels, ranging from isolated proteins and protein assemblies to highly complex protein mixtures, such as cell lysates and intact cells.
Erwin Schoof, Technical University of Denmark
Title: Leveraging latest-generation data acquisition methods for improving single cell proteomics
Julia Cunha, Butantan Institute, São Paulo, Brazil
Title: Unrevealing the epigenetics of a protozoa parasite by multi-omics approaches
Summary: Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. In this scenario, the role of epigenetic changes would be questionable. However, these parasites harbour many epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages. Here, we will show the global dynamics of chromatin proteomics and histone post-translational modifications along Trypanosoma cruzi life and cell cycle. Epigenomic (FAIRE-seq, MNase-seq, Chip-seq, and Hi-C data) and transcriptomic (mature and nascent RNA-seq) approaches also highlighted the impact of epigenetic changes even in a scenario commanded by posttranscriptional control. Finally, proteomic strategies to detect interaction partners of critical epigenetic players and techniques to recover loci-specific proteins will be discussed. Supported by FAPESP, Serrapilheira and CAPES.
Lorena Martin-Jaular, Institute Curie, Paris, France
Title: Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection.
Summary: Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.
Mehdi Bouhaddou, UCSF, San Francisco, USA
Title: Proteomics and bioinformatics of virus-host signaling networks and protein complexes
Summary: Viruses must interact with host molecular machinery to replicate and transmit. Proteomics and bioinformatics approaches allow us to globally quantify and interpret the host response to infection, and subsequently pin down mechanisms of viral pathogenesis. Previously, we have globally profiled changes in viral and host protein levels and post-translational modifications (e.g. phosphorylation) during infection. We have additionally profiled virus-host protein complexes using Affinity Purification Mass Spectrometry (APMS). We recently applied our technologies to studying SARS-CoV-2. In a recent study, we discovered SARS-CoV-2 variants of concern (VOC) differ in viral RNA and protein expression, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An integrative bioinformatics analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. Our findings describe how SARS-CoV-2 has evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. Our approaches are virus- and disease-agnostic, with applications to a range of biological areas.
Rebeca Kawahara, Macquarie University, Sydney, Australia
Title: Glycoproteome signatures associated with cancer progression
Summary: Glycosylation is a prevalent and complex type of protein modification central to many biological processes underpinning cancer development and progression. Facilitated by advances in separation sciences, mass spectrometry and informatics, glycoproteomics is now ready to profile and generate new insights into the dynamics and complexity of the heterogenous cancer glycoproteome and discover new glycosignatures associated with cancer progression. In this seminar, I will 1) briefly highlight new mass spectrometry-based strategies recently developed by our group to study protein glycosylation with site-, protein- and cell-specific resolution at the system-wide level and 2) provide recent examples of how glycoproteomics can be used to discover glycosignatures in cancer and 3) understand glycosylation-related mechanisms within the complex tumour microenviroment. These efforts improve our understanding of the fundamental glycobiology underpinning cancer progression and open new exciting opportunities for diagnostic, prognostic and therapeutic applications.
Tony Wyss-Coray, Stanford University School of Medicine, USA
Title: Plasma proteomics to study aging and neurodegeneration
Summary: Aging results in loss of function and disease across most tissues and organs. This is accompanied by dramatic changes in the expression of genes and the concentration of molecules across all chemical classes. Technological advances have enabled biologist to measure ever larger numbers of these molecules across an organism and derive unprecedented new information about physiological and pathophysiological states of cells and organs including aging. Our studies have focused on brain aging which leads to cognitive decline and is the main risk factor for sporadic forms of neurodegenerative diseases including Alzheimer’s disease. While brain cell- and tissue-intrinsic factors are likely key determinants of the aging process recent studies document a remarkable susceptibility of the brain to circulatory factors. Thus, blood borne factors from young mice or humans are sufficient to slow aspects of brain aging and improve cognitive function in old mice and, vice versa, factors from old mice are detrimental for young mice and impair cognition. We found evidence that plasma proteins are taken up broadly into the young brain through receptor mediated transport which declines with aging. At the same time, brain derived proteins are detectable in plasma allowing us to measure physiological changes linked to brain aging in plasma. Likewise, proteins derived from every other organ and cell type can be quantified in plasma enabling new ways to monitor physiology and aging towards personalized monitoring of health and disease and the identification of new drug targets.
Waters – Roy Martin
Title: High-Definition (HD) Proteomics Strategies apply highly innovative technologies to comprehensively characterize the proteome
Analitica – José Felipe Lugão
Title: Novo Orbitrap Ascend