M 05
Comparison of HPLC and Capillary Electrophoresis for Hydrogen Exchange Mass Spectrometry

Presenting author:

Uppsala universitet, Pharmaceutical Biosciences, husargatan 3, 751 24 Uppsala [SE], jordan.aerts@uu.se

Author(s):
Jordan Aerts, Jonathan Zöller, Julian Langer, Erik Jansson

Hydrogen deuterium exchange mass spectrometry (HDX-MS) has been a valuable tool for structural proteomics studies for more than 30 years. Labeling of proteins with deuterium in solution is a straightforward experiment, but downstream sample handling steps should be conducted under quench conditions (low temperature and pH) to maximize the structural information obtained from protein and peptide measurement. Traditional HDX-MS workflows utilize low-temperature liquid chromatography (LC) with short gradients for peptide separations. However, operating an LC system at low temperatures generally suffers from increased sample carry-over, and the need for expensive system components. Cold capillary electrophoresis (CE) separations offer a low cost method of separating peptides at quench conditions for HDX-MS workflows. Here we present a direct comparison at the peptide level using bovine hemoglobin analyzed on both a laboratory-built CE platform and a fully automated Waters HDX-2 system, both measured with a Waters Synapt G2-Si. The quenched, and digested protein (10,000 fmol on column for LC, 50 fmol on capillary for CE) was measured after labeling in D₂O for 0, 50, 500, 5000, and 50,000 s. Preliminary results demonstrate similar deuterium uptake curves for proteolytic peptides detected across both separation methods, at significantly lower sample amounts. These findings validate the use of cold capillary electrophoresis as an alternative to HPLC in HDX-MS workflows.

Short talk 5
Investigation of the human lysosomal proteome by targeted proteomics

Presenting author:

University of Bonn, , Nussallee11, 53115 Bonn [DE], darora@uni-bonn.de

Author(s):
Dhriti Arora, Stephanie Kaspar-Schoenefeld, Andreas Schmidt, Dominic Winter

Lysosomes, the main lytic organelles of mammalian cells, play a vital role in cellular homeostasis. This is facilitated by ~340 lysosomal-related proteins whose loss of function can result in a variety of disorders. To study diseases and cellular processes related to lysosomes, reproducible quantification of these proteins is crucial. However, the low abundance of the organelle makes it difficult to quantify these proteins using untargeted proteomics. Therefore, DIA and targeted approaches such as PRM are the most effective methods for the detection of lysosomal proteins. In this study, we investigated the lysosomal proteome of four human cell lines by dia-PASEF, merging the benefits of DIA with the advantages of ion mobility in proteomics. To investigate the coverage of lysosomal proteins in whole-cell lysates and lysosome-enriched fractions, we used a targeted data processing library consisting of 297 manually selected lysosomal proteins. To assess the quantitative performance a lysosome-enriched sample was spiked into the whole cell lysate to simulate the constitutive upregulation of lysosomal proteins. A total of 165 lysosomal proteins showed significantly higher abundance indicating that dia-PASEF is well suited for analysing the lysosomal proteome, providing both good coverage and quantitative reproducibility of the targeted lysosomal proteins. Finally, we developed prm-PASEF assays based on our dia-PASEF analyses to enable targeted analysis of lysosomal proteins

G 11
Protein Synthesis in Autism Spectrum Disorder

Presenting author:

Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany, Proteomics , Blankestr 8A, 13403 Berlin [DE], jose.astorga@mdc-berlin.de

Author(s):
Jose Astorga

Autism spectrum disorder (ASD) is a complex, lifelong and highly prevalent neurodevelopmental disorder. ASD exhibits significant heterogeneity in genotypes and phenotypes. In recent years, dysregulation of protein synthesis has emerged as a convergent mechanism underlying ASD. The goal of this project is to better understand the pathophysiology of ASD by investigating clinically relevant mutations associated with protein synthesis. To achieve this goal, ASD-related mutations were introduced into induced pluripotent stem cells (iPSCs) derived from healthy donors. These iPSCs were then differentiated into neurons and their proteome were analysed by mass spectrometry. Given the highly polarised nature of neurons, investigating the local proteome of these cells is of great interest. For this purpose, neurons will be cultured using inserts that allow the soma to be separated from the neurites, which facilitates the analysis of the cell's local proteome. In addition, a pulsed stable isotope stable amino acid labelling in cell culture (SILAC) technique will be used to quantify changes in protein synthesis. Once these methodologies are established, cell lines carrying PTEN and TSC2 mutations, which are clinically relevant in ASD, will be incorporated into the study. Consequently, the results of this research are expected to elucidate the molecular underpinnings of ASD, encompassing not only case-specific mutations, but also alterations shared between different ASD-related mutations.

G 04
Autoprot: A Modular Package for Processing, Analysis and Visualization of Complex Proteomics Data in Python

Presenting author:

Würzburg University, Chair of Biochemistry II, Am Hubland, 97074 Würzburg [DE], julian.bender@uni-wuerzburg.de

Author(s):
Julian Bender, Wignand W. D. Mühlhäuser, Johannes P. Zimmermann, Friedel Drepper, Bettina Warscheid

The rising complexity of mass spectrometry (MS) data sets in proteomics research requires standardized and reliable data analysis workflows. Python-based software tools, particularly jupyter notebooks, provide a simple yet powerful solution for this. However, there is only a limited repertoire of Python software available for standardised and easy-to-use MS data analysis. This software is often restricted to algorithms developed in Python while excluding existing and well-tested software developed in other programming languages, such as R. Furthermore, current Python software frequently lacks interactive data visualization for improved and convenient exploratory inquiries and sharing of results with collaboration partners. We developed autoprot, a Python module for analysing MS-based proteomics search results generated with the MaxQuant software. Autoprot offers access to functions in Python and R for statistical testing and data transformation. Furthermore, it generates dynamic javascript-based charts that can be integrated into interactive web applications. We show the application of autoprot using publicly available MS datasets, highlight functions of the submodules for data preprocessing, analysis and visualisation and showcase interactive plots generated with the software. In summary, autoprot provides standardised, fast, and reliable proteomics data analysis while ensuring a high customisability needed to tailor the analysis pipeline to specific experimental strategies.

G 14
Using quantitative proteomics to uncover ribosome heterogeneity in neurons

Presenting author:

Max Planck Institute for Brain Research, Department of Synaptic Plasticity, Max-von-Laue Str. 4, 60438 Frankfurt am Main [DE], ashley.bourke@brain.mpg.de

Author(s):
Ashley Bourke, Kristina Desch, Claudia Fusco, Sara Mota, Julian Langer, Erin Schuman

Customized remodeling of synaptic proteomes is essential for proper neuronal function. During brain development and plasticity, local protein synthesis is differentially regulated in individual synaptic compartments to control synapse formation and strength (Bernard et al., 2022; Hafner et al., 2019), however the repertoire of molecular mechanisms used is not well understood. A promising mechanism for sculpting synapse-specific proteomes is selective mRNA translation by 'specialized ribosomes' - ribosomes with different subunit compositions or associated proteins. This possibility is supported by recent findings of synaptic mRNA translation on 80S monosomes (Biever et al., 2020) and context-dependent ribosome remodeling in dendrites (Fusco et al., 2021), however the extent to which neuronal ribosomes are compositionally distinct remains unknown. Here, we use two MS-based approaches to identify ribosome-associated proteins at specific subcellular regions and across ribosomal subcomplexes. In the first approach, we combine proximity labeling, ribosome purification, and label-free quantification to map the protein interactomes of ribosomes in the nucleus (nuclear membrane), cytoplasm, and dendritic spines of rat hippocampal neurons. In the second approach, we use polysome proteome profiling (Imami et al., 2018), which couples sucrose gradient fractionation with SILAC-based proteomics, to identify the interactors of monosomes, polysomes, and other ribosomal subcomplexes.

G 18
The Hidden Role of Reactive Oxygen Species in Modulating Mitochondrial-Processing Peptidase

Presenting author:

Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], bozkurt@med.uni-frankfurt.de

Author(s):
Süleyman Bozkurt, Doha Boutguetait, FX Reymond Sutandy, Christian Münch

Most mitochondrial proteins, encoded in the nucleus, require accurate translocation into mitochondria. The mitochondrial-processing peptidase (MPP), critical for precursor protein processing, ensures their proper function. Any disruption in this process can lead to protein dysfunction, causing diseases. Reactive Oxygen Species (ROS), including H2O2, are small, reactive molecules produced within cells, particularly in mitochondria. ROS regulate growth and stress response. Despite being a ROS, H2O2 is a vital cellular signaling molecule due to its permeability through cell membranes. We employed a peroxisomal enzyme, D-alanine oxidase (DAO), to study ROS signaling. DAO catalyzes D-amino acids into pyruvate, ammonia, and H2O2. We targeted DAO to mitochondrial matrix, and conducted biochemical experiments, including proteomics. H2O2 production increased with D-alanine treatment, verified with Hyper7 probe. Longer treatment caused mitochondrial stress; reducing MMP, protein import, leading to precursor protein accumulation and OMA1 activation. Shorter treatment induced precursor protein accumulation in the mitochondrial matrix, suggesting the malfunction of MPP. In conclusion, mitochondrial proteins must be accurately imported and processed by MPP for proper function. Disruptions to MPP can lead to unprocessed protein accumulation in mitochondria, triggering serious cellular complications and potentially contributing to diseases.

M 11
Let’s make it clear: systematic exploration of mitochondrial DNA-/RNA-protein complexes by complexome profiling

Presenting author:

Goethe Universität Frankfurt, Institute for Cardiovascular Physiology, Theodor-Stern-Kai 7, Haus 26, 5th floor, 60590 Frankfurt am Main [DE], alfredbiomed@gmail.com

Author(s):
Alfredo Cabrera-Orefice, Alisa Potter, Johannes N. Spelbrink

To synthesize the mtDNA-encoded proteins, both strands of the circular mtDNA are transcribed into polycistronic RNAs, which are processed and maturated generating tRNAs, rRNAs and mRNAs for mitochondrial translation. The mtDNA replication and gene expression machinery are tightly regulated by specific sets of nuclear-encoded proteins. Although the roles of the major mitochondrial nucleic acid-interacting proteins have been described, a lot of interactors remain unverified or unknown. We have improved native gel electrophoresis-based complexome profiling (CP) for examination of mitochondrial DNA-/RNA-protein complexes. Our adaptations enabled the systematic exploration of mtDNA- and RNA-protein interactions in human mitochondria, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome. To illustrate the applicability of our approach, we performed a proof-of-principle experiment using inhibition and recovery of transcription with a transient ethidium bromide treatment, identified and validated many of the known mitochondrial protein-RNA interactions involved in, for instance, mitoribosome biogenesis. Thus, our method not only helps validate and unveil proteins involved in mitochondrial DNA-/RNA-related processes, but also offers a convenient and systematic way to analyse these interactions that can be virtually applied to investigate any kind of nucleic acid-protein complexes.

G13
A study on small proteins present in terminal cytochrome oxidases

Presenting author:

Max-Planck Institut für Biophysik, Mass Spectrometry and Proteomics, Max-von-Laue Straße 3, 60438 Frankfurt [DE], pedrofrancobh@gmail.com

Author(s):
Pedro Henrique Cavalcanti Franco, Rilee Zeinert, Imke Wüllenweber, Gisela Storz, Julian Langer

Small proteins, < 50 amino acids in length, have been shown to regulate cellular processes such as antibiotic resistance and cell development. Despite their perspective as therapeutics, their characterization remains limited due to insufficient gene annotation and challenges associated with their characterization. In E. coli, CydH (29 aa) and CydX (37 aa) bind the cytochrome bd-I oxidase and AppX (30 aa) binds the cytochrome bd-II oxidase. However, their specific function within the complexes and potential interaction with additional complexes remain unclear. In this study we investigated the binding partners of CydX and CydH during aerobic and anaerobic growth. To identify conditions for interaction studies we monitored expression levels in these conditions and examined the impact of their deletion on E. coli growth. We find that all three small proteins are induced in anaerobic conditions, being CydX expression the most abundant relative to the others. We performed immunoprecipitation assays to look for interacting partners of CydH and CydX in aerobic vs anaerobic growth and current work is aimed at globally identifying putative partners using LC-MS/MS. The results thus far suggest these small proteins might be most important during anaerobic growth or transitions between aerobic and anaerobic growth, conditions for which the roles of small proteins have not been explored.

G 25
Investigating novel functions of Rab24 in mitochondrial fission and protein secretion

Presenting author:

LMU, Munich Cluster of system Neurology, Feodor-Lynen Str. 17, 81377 Munich [DE], rahulchakraborty725@gmail.com

Author(s):
Rahul Chakraborty, Syed Qaaifah Gillani, Anja Zeigerer, Christian Behrends

Globally, the incidence of non-alcoholic fatty liver disease (NAFLD), a crucial factor in type 2 diabetes caused by obesity, is rising. More severe liver damage, such as cirrhosis and hepatocellular carcinoma, arise because there are currently no robust treatment options. Here, we explore an unanticipated role for the small Rab GTPase Rab24, an intracellular trafficking regulator, in mitochondrial fission and activation, which directly affects hepatic and systemic energy homeostasis. RAB24 has previously been demonstrated to be significantly elevated in livers of obese individuals with NAFLD and to have a strong positive correlation with increased body fat in humans. This atypical GTPase has recently been discovered as a novel interactor of mitochondrial fission protein Fis1 in the liver. Split GFP protein complementation assays and APEX2-based proximity labeling approaches are now employed to identify Rab24-Fis1 regulating and scaffolding proteins. In both strategies, candidate interacting or neighboring proteins are enriched by affinity purification and subsequently identified by mass spectrometry. Complementarily, we are performing whole cell protein abundance profiling in conditional Rab24 or Fis1 knockout cells using DDA- and DIA-based mass spectrometry to uncover factors that operate downstream of the Rab24-Fis1 interaction. Together, these efforts will help to decipher the functional role of Rab24 in the context of Fis1.

G 24
Proteomic profiling of sex- and oestrus-cycle specific changes in the midbrain

Presenting author:

Max-Planck-Institute for Brain Research, Synaptic Plasticity, Max-von-Laue-Straße 4, 60438 Frankfurt [DE], kristina.desch@brain.mpg.de

Author(s):
Kristina Desch, Elena Kutsarova, Petros Chalas, Genesis Rosiles, Vanessa Stempel, Julian Langer

While instinctive behaviors are evolutionarily conserved, they can be adapted to different environments and internal states of animals. Despite its involvement in many of these behaviors, the midbrain periaqueductal gray (PAG) has been mostly considered a simple relay station. However, expression of certain candidate proteins suggests that it may be able to confer behavioral flexibility. To understand how individual behavior is modulated in the PAG and if it allows for context-dependent adaptations, we characterized its proteomic composition and investigated possible sex- and estrous-cycle-specific changes using DIA-LC-MS. Initial results revealed comprehensive proteomic coverage with ~7,300 proteins per sample. Proteins associated with synaptic plasticity were abundantly identified suggesting that the PAG has the potential to undergo plasticity-induced changes. While the overall proteomic composition was highly similar among all animals, several extracellular matrix proteins implicated in Alzheimer's disease and synaptic remodeling showed differential abundance between male and female mice. Interestingly, no reliable changes during different stages of the estrous cycle were observed. In conclusion, the proteomic compositions of the male and female mouse PAG are largely similar with a few proteins showing differential abundance. For further characterization and to overcome dilution effects from bulk tissue analysis, future studies may require cell-type-specific labeling.

O 01
Why do mitochondria still contain a genome? Mechanistic insights from allotopically expressed proteins

Presenting author:

RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], ecker@rhrk.uni-kl.de

Author(s):
Anna-Lena Ecker, Johannes M. Herrmann

Mitochondria are essential organelles of eukaryotic cells. They consist of hundreds of nuclear encoded proteins, but also harbor a small genome as a remnant of a bacterial ancestor. Mitochondrial genomes encode a small number of very hydrophobic proteins. Why the genes of these proteins were not transferred into the nucleus is not well understood. To elucidate the molecular consequences of such mitochondria-to-nucleus gene transfer reactions, we allotopically expressed the model proteins Cox3 and Atp6 with mitochondrial targeting sequences in the cytosol of yeast cells. The fusion proteins are not imported into mitochondria but rather accumulate on the cytosolic surface of the outer membrane translocase. The highly hydrophobic character of these proteins presumably prevents efficient translocation through the TOM complex. These stalled translocation intermediates are efficiently removed by proteolysis, specifically by components of the cytosolic ubiquitin-proteasome system (UPS). Mutants in the UPS which prevent the efficient degradation of these proteins lead to growth defects and induce cell death. Thus, the protein quality system on the mitochondrial surface is important for cellular functionality, however, it prevents the productive gene transfer from mitochondria to the nucleus and forces eukaryotic cells to maintain the genes of a core set of highly aggregation-prone proteins.

G 02
The interplay of posttranslational protein modifications in Arabidopsis leaves during photosynthesis induction

Presenting author:

University of Muenster, Institute of Plant Biology and Biotechnology, Schlossplatz 7, 48149 Muenster [DE], juergen.eirich@wwu.de

Author(s):
Juergen Eirich, Jonas Giese, Iris Finkemeier

Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM). A global picture of the light-dependent PTMome dynamics was lacking so far. Here we investigated the light-dependent proteome changes in Arabidopsis leaves in a time-resolved manner to dissect global phosphorylation, lysine acetylation, and cysteine-based redox switches using different quantification strategies, including DiMethyl- and iodoTMT labeling. Of over 24,000 PTM sites that were detected on an Orbitrap Q Exactive HF, more than 1,700 were changed during the transition from dark to light. While the first changes, as measured 5 min after the onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveals connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insights into the extent by which photosynthesis re-programs global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.

G 22
Quantitative Secretome Kinetics

Presenting author:

Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], fehmer@med.uni-frankfurt.de

Author(s):
Martin Fehmer

Secreted proteins play a central role in coordinating both basic biological functions such as cell growth, division and differentiation as well as complex cellular programs including apoptosis and signaling. It is estimated that about 15% of the human genome encode factors that are putatively secreted, with about roughly a third of these factors acting locally in a tissue- or microenvironment-specific manner. Alterations in the cellular secretome composition have been associated with several malignancies, including the development of chemoresistance, the progression and modulation of infectious diseases, as well as mast cell dysfunction. Mass spectrometry-based proteomics have been successfully integrated into mapping the cellular secretome, providing insights into both the fundamental composition as well as disease associated changes of the extracellular environment. These studies are comprised of either stand-alone proteomic datasets or encompass a comparative analysis between one or several conditions to a control without assessing the accumulation of secretory proteins in a time-resolved manner. Using a SILAC-TMT-based approach following the mePROD method developed by Klann et al. in 2020, we set out to establish cellular secretion kinetics across the secretome. We then strive to apply this method towards the investigation of secretory disease models as well as delineating the route different components may take along both canonical and non-canonical secretory pathways.

Short talk 4
A toolbox for systematic discovery of stable and transient protein interactors in baker’s yeast

Presenting author:

Weizmann Institute of Science, Molecular Genetics, 234 Herzl Street, 7610001 Rehovot [IL], emma.fenech@weizmann.ac.il

Author(s):
Emma Fenech, Maya Schuldiner

Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries; some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.

Sept. 6, 9:00
Understanding the tumor microenvironment through high-sensitivity MS-based proteomics

Presenting author:

Weizmann Institute of Science, , Herzel 234, 7610001 Rehovot [IL], tami.geiger@weizmann.ac.il

Author(s):
Tami Geiger, Mariya Mardamshina, Shiri Karagach, Vishnu Mohan

Cancer heterogeneity presents a significant challenge to effective treatment strategies. Genetic variations and cellular interactions within the tumor microenvironment (TME) contribute to the diverse molecular characteristics observed among different tumor clones. Understanding the functional proteomic layer of tumor subpopulations and their interactions with the microenvironment is crucial. In this study, we integrated mass spectrometry-based proteomics with spatial multiplexed imaging of cells from the TME to unravel the functional proteomic layer of breast cancer heterogeneity. Our approach combined clinical sample analysis, multilayer tissue imaging, and deep learning-based image processing to identify novel regulators of cancer phenotypes. Analyzing hundreds of breast cancer tumor regions, we discovered associations between clinical parameters, protein networks, and intra-tumor heterogeneity. Proteins related to cell adhesion and interactions with the immune system exhibited the highest variability, while proteins related to cell proliferation remained constant. Furthermore, our analyses highlighted the proteomic impact of distance from blood vessels, tumor center, and immune cells, including T-cells and macrophages. By integrating mass spectrometry-based proteomics and spatial multiplexed imaging, we provide valuable insights into the functional proteomic layer of breast cancer heterogeneity, offering new avenues for targeted therapies and personalized medicine.

M 09
Combining Data Independent Acquisition with Spike-in SILAC (DIA-SiS)

Presenting author:

MDC Berlin, , Robert-Rössle-Straße 10, 13125 Berlin [DE], maximilian.gerwien@mdc-berlin.de

Author(s):
Maximilian Gerwien, Anna Sophie Welter

SILAC-based quantification has been extensively applied in DDA proteomics for many years due to its superb quantitative performance. However, SILAC involves the metabolic labelling of cultured cells. Since this is not always possible or convenient (e.g., clinical samples), a previously prepared SILAC spike-in can be employed. Recently, DIA proteomics became more popular. It offers unbiased and reproducible profiling of peptides over a broad dynamic range. To combine the merits of spike-in SILAC with DIA proteomics, we devised DIA spike-in SILAC (DIA-SiS). As a stable isotope labelling method, it is precise and accurate. As a spike-in method, it is almost as easy and fast to use as a label-free approach. And, as a DIA method, it benefits from the unbiased and comprehensive profiling of precursor ions. To assess the quantitative performance (number of identifications, accuracy and precision) of DIA-SiS compared to label-free DIA, we created a benchmark dataset with known quantities. Here, we show that DIA-SiS improves the identification and quantification of precursors and proteins of low-input samples. From 10 ng human proteome digest, we quantify >2000 proteins with spike-in compared to ca. 1000 proteins without spike-in. Overall, coefficients of variation of proteins are lower with the spike-in. In summary, DIA-SiS offers improved identification and quantification of low-abundant samples compared to label-free DIA.

G 12
Nuclear localization of non-imported mitochondrial proteins modulates epigenetic landscape

Presenting author:

RPTU Kaiserslautern-Landau, DEPARTMENT OF CELL BIOLOGY, kohlenhofstrasse 3, 67663 kaiserslautern [DE], nikita.gupta@rhrk.uni-kl.de

Author(s):
Nikita Gupta, Johannes Herrmann

Most of the mitochondrial proteins are synthesized in the cytosol and are translocated to mitochondria via the mitochondrial import machinery. However, under import failure, the non-imported mitochondrial precursor proteins get accumulated in many regions of the cell, with the nucleus being one of the key locations for quality control. Still, it remains unclear what drives these non-imported mitochondrial precursor proteins to the nucleus and whether these mitoproteins exhibit any metabolic or regulatory function in the nucleus. To elucidate the consequences of mitochondrial import failure in the epigenetic landscape of the cell, we then investigated histone synthesis under the expression of a clogger protein. To our surprise, we observed that the synthesis of the histone gene is strongly repressed under import failure hinting towards a potential role of non-imported mitochondrial proteins which accumulate in the nucleus in regulating the epigenetic landscape of the cell. The aim of my study is to characterize the functional role of non-imported mitochondrial proteins which localize to the nucleus upon mitochondrial dysfunction.

C 01
Loss of nuclear pore complex function and cellular compartmentalization in the steroid resistant nephrotic syndrome

Presenting author:

Uniklinik Aachen, Biochemie (AG Antonin), Pauwelsstraße 30, 52074 Aachen [DE], mhamed@ukaachen.de

Author(s):
Mohamed Ismail Hamed

Focal segmental glomerulosclerosis (FSGS) is a progressive pathology with gradual loss of kidney function and end-stage kidney failure. FSGS is characterized by loss of podocyte function, a cell type that forms the kidneys’ filtration barrier. Podocytes are post-mitotic cells with no proliferative capacity which accordingly reside within the kidney for life time. As a result, proteins and protein complexes with long residual times are prone to insults such as mutations if dedicated repair mechanisms are lacking. Nuclear pore complexes (NPCs) are a multi-protein complexes integrated within the nuclear envelope (NE). The NE separates the cytoplasmic and nuclear compartments and NPCs act as the transport gates. Mutations in a number of NPC proteins cause the steroid resistant nephrotic syndrome, a FSGS with childhood-onset, characterized by podocyte and kidney function loss otherwise typically seen in older patients. We hypothesize that these mutations weaken the NPCs and lead to a progressive loss of cellular compartmentalization, which in healthy persons is only observed much later in life. Using mass-spectroscopy and immunofluorescence microscopy, we characterize compartmentalization loss in podocytes of aging mice and define specific markers to follow disease progression. Furthermore, we have established cellular assays where loss of compartmentalization by NPC defects can be recapitulated and which are currently used for compound screening to retain proper NPC function.

S 04 & Short talk 1
SNARE complex regulation by Complexin-1 - a structural mass spectrometry study

Presenting author:

Johannes Gutenberg University Mainz, Chemistry - Biochemistry, Hanns-Dieter-Hüsch-Weg 17, 55128 Mainz [DE], julia.hesselbarth@uni-mainz.de

Author(s):
Julia Hesselbarth, Carla Schmidt

Signal transmission between neurons is mediated by the SNARE complex that is responsible for fusion of synaptic vesicles with the presynaptic membrane. This ternary complex assembles from vesicular Synaptobrevin-2 as well as SNAP25 and Syntaxin-1A, which are both anchored to the presynaptic membrane. SNAP25 contributes two alpha-helices and Syntaxin-1A and Synaptobrevin-2 both contribute one alpha-helix forming a stable four-helix bundle. The SNARE assembly is a highly regulated process involving Complexin-1, which is known to bind a groove formed by Synaptobrevin-2 and Syntaxin-1A, however, the regulatory mechanism is largely unknown. Using native mass spectrometry, we first investigated interactions of Complexin-1 with individual SNAREs and binary SNARE complexes to elucidate a regulatory function in early states of SNARE assembly. While Complexin-1 does not interact with individual SNAREs or binary complexes similar to the SNAP25:Syntaxin-1A complex, Complexin-1 binding stabilizes the Syntaxin-1A:Synaptobrevin-2 interface leading to formation of a complex that imitates SNARE complex stoichiometry. Following incubation of all three SNAREs, formation of the SNARE complex and oligomers thereof was observed. Addition of Complexin-1 disassembled these oligomers indicating an inhibitory role for SNARE oligomerisation. Specific interaction sites of Complexin-1 within this assembly were further explored by chemical cross-linking providing a model of the SNARE:Complexin-1 complex.

M 12
De novo Protein Interactome Profiling of Small Molecule and Antisense Oligonucleotide Drugs

Presenting author:

Eberhard Karls Universität Tübingen, Interfaculty Institute of Biochemistry, Auf der Morgenstelle 15, 72076 Tübingen [DE], daniel.hofacker@uni-tuebingen.de

Author(s):
Daniel Hofacker, Alfred Hanswillemenke, Thorsten Stafforst

Protein interactions determine the pharmacological properties of a drug including toxicity, immunogenicity, efficacy, metabolism, and adverse effects. In this yet unpublished work, we present a toolbox to comprehensively identify the protein interactome for several drug types de novo in living cells. This includes small molecule drugs as well as antisense oligonucleotide (ASO) drugs, which have gained notable clinical relevance in the past few years. The interactome is identified by covalent recruitment of a biotin ligase to the drug of interest followed by targeted protein biotinylation, and biotin enrichment coupled to MS². Compared to pulldown-based approaches that identify RNA drug interactions in vitro, our novel approach can distinguish between different ASO chemistries at pharmacologically effective intracellular concentrations, and furthermore, mesoscale changes in the RNA/protein interactome in response to stress were identified. We further extended the method to discover the interactome of endogenous lncRNAs that recruit proteins to specific nuclear condensates. In contrast to established protocols, our assay does not need any genetic modification of the cells, uses simple probes, and requires up to 100-fold less input material than in vitro pulldown-based methods, making it suitable for hard-to-transfect and non-dividing cells. Overall, this powerful tool helps to unravel the intricate RNA/protein interplay and offers valuable insights into RNA drugs and RNA biology.

G 03
Gliflozin drug class and its effect on the proteome of cardiomyocytes

Presenting author:

, , Albrechtsbergergasse 19/2, 1120 Wien [AT], julia.hoehlschen@tuwien.ac.at

Author(s):
Julia Höhlschen, Tamara Tomin, Ruth Birner-Grünberger

Death from cardiovascular (CV) diseases is the most relevant macrovascular complication in type 2 diabetes. A new drug class that offers cardioprotective properties are sodium-glucose co-transporter-2 (SGLT-2) inhibitors, initially used for the treatment of type 2 diabetes. Meanwhile trials have shown that these properties are beyond the effect of lowering glucose concentrations in the blood. Therefore, their application in non-diabetic patients suffering from heart diseases has recently been approved. This project aims to identify the not yet understood mechanisms leading to the observed cardioprotective effects. In a first experiment I applied redox-proteomics to check if the drugs show antioxidative effects on a differentiated, human cardiomyocyte cell line (AC16) by mimicking disruption of oxygen supply (similar to heart failure): 1% oxygen (hypoxia), 21% oxygen (Control) and reperfusion injury (1% followed by re-oxygenation at 21% oxygen), as oxidative stress is one potential cause of heart failure.

O 04
Akt isoforms differentially affect Rho signaling pathways in H23 non-small cell lung carcinoma cells

Presenting author:

Justus Liebig University Giessen, Signal Transduction of Cellular Motility, Aulweg 128, 35392 Giessen [DE], Bahareh.Jooyeh@innere.med.uni-giessen.de

Author(s):
Bahareh Jooyeh, Stefanie Wirth, Manfred Jücker, Andre Menke, Klaudia Giehl

Studies in our group showed that oncogenic K-Ras regulates cell migration of carcinoma cells by modulating the PI3-K/Akt pathway and expression of the three Akt isoforms. In this study, H23 lung carcinoma cells harboring K-Ras(G12C), were used to elucidate a possible cross-talk between Akt and Rho signaling. Stable lentiviral transduced Akt isoform-specific knockdown (kd) H23 cell clones were generated to investigate the impact of each Akt isoform. Additionally, different pharmacological inhibitors for Akt, PI3-K, RhoA, and ROCK were used. MK-2206, a pan-Akt inhibitor, downregulated the phosphorylation of all Akt isoforms, but it did not affect the phosphorylation of the ribosomal protein S6. LY-294002 inhibited Akt and S6 phosphorylation. Inhibition of RhoA by Rhosin resulted in decreased phosphorylation of Akt in all analyzed cell clones, without affecting S6. However, we observed no changes on Akt and S6 by Y-27632, ROCK inhibitor. Western blot analyses revealed that RhoA protein expression was upregulated in Akt2-kd H23 cells, whereas Akt1-kd cells displayed an increased expression of Rac1b. The upregulated RhoA expression resulted in a higher content of active RhoA in Akt2-kd cells. Furthermore, knockdown of Akt1 and Akt2 led to an upregulation of cofilin and an increase in the phosphorylated form. With our findings, we aim to elucidate the intracellular communication between K-Ras/Akt and Rho signaling pathways and their impact on cell migration and metastasis.

M 04
Improved dia-PASEF isolation window schemes for proteomics measurements

Presenting author:

Bruker Daltonics GmbH & Co KG, , Fahrenheitstraße 4, 28359 Bremen [DE], stephanie.kaspar-schoenefeld@bruker.com

Author(s):
Markus Lubeck, Stephanie Kaspar-Schoenefeld, Christoph Krisp, Andreas Schmidt, Gary Kruppa

DIA is widely used for proteomics as it promises reproducible and accurate protein identification and quantitation. dia-PASEF is both more sensitive and selective than traditional DIA approaches as it combines the advantages of DIA with the inherent ion-usage efficiency of PASEF. Making use of the correlation of molecular weight and CCS coded information, dia-PASEF enables highly confident identification. The two-dimensional mass and mobility space enables method creation with extensively different window schemes. Here, a variety of fixed-width as well as more advanced window schemes were evaluated. Dilution series of tryptic digests from human cell lines were separated using different nanoLC gradients. Different isolation windows widths were compared to more sophisticated approaches like schemes with variable window widths based on precursor density (py_diAID). Data were processed using Spectronaut 18 (Biognosys). In the presented study, we limited the dia-PASEF windows to the mass and mobility range of highest precursor density. For sample amounts in the 10-50 ng range identifications were remarkable similar among the different tested acquisition schemes. Lower sample amounts benefit from a lower number of broader windows. For higher sample loads, acquisition schemes of more narrow isolation windows resulted in improved identifications due to their higher specificity. Optimal methods for a broad range of sample amounts and gradient lengths could be determined.

G 15
Using proteomics to characterize RNF213- a unique AAA+ ATPase and E3 ligase

Presenting author:

Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], kohli@med.uni-frankfurt.de

Author(s):
Aneesha Kohli, Christian Münch

Moyamoya disease associated protein RNF213 is a large AAA+ ATPase and an E3 ligase. Recently, it was reported to play a role in xenophagy, cancer and lipid metabolism apart from its previously briefly described role in immune response, inflammation and angiogenesis. Despite the rising interest, its role in basal and inflammatory states remains largely unknown. We aim to use proteomics as a way of characterizing the functional role of RNF213 in basal and stressed states wherein for the latter we employ IFNγ, a known inducer of RNF213. In this regard, we have thus far, applied global proteomics approach to identify the key proteins and the associated pathways that are modulated in the absence of RNF213 using knockdown assays. We have used interactomics to identify its direct or complex associated interaction partners as well as E3 targets. Our proteomic analyses provide an insight into the role of RNF213 in immune response as reported earlier but also in other pathways such as mitochondrial biogenesis and ribosomal machinery, protein transport and rather crucially, also in heme biogenesis and ferroptosis. Next, we aim to further our findings by complementing our work with ubiquitinomics to correctly identify the E3 targets and sites of its associated activity on the target proteins in the future and dive further into its mechanistic role in ferroptosis.

G 17
Single-cell multiomics on brain organoid models of autism spectrum disorder

Presenting author:

Max Delbrück Center (MDC), , Robert-Rössle Str. 10, 13125 Berlin [DE], marianna.kokoli@mdc-berlin.de

Author(s):
Marianna Kokoli, Matthias Selbach

Autism Spectrum Disorder (ASD) is one of the most complex neurodevelopmental disorders, characterized by atypical social, behavioral and cognitive function. ASD exhibits remarkable heterogeneity in terms of genotypes and phenotypes, yet currently lacks early diagnostic methods and effective treatments. In recent years, dysregulated protein synthesis has emerged as a prominent feature of ASD. This project aims to shed light on the molecular mechanisms underlying ASD with respect to clinically-relevant mutations in FMR1, PTEN and TSC2 genes which affect protein synthesis. In order to better recapitulate and model neurodevelopment during ASD, we plan to generate brain cerebral and micropatterned organoids from wildtype induced pluripotent stem cells (iPSCs) and iPSCs harboring the aforementioned ASD-related mutations. A multiomics approach of transcriptomics and proteomics both at the bulk and the single-cell level will be employed so as to investigate the correlation between mRNA and protein levels, as well as to identify alterations in protein translation over time. Overall, with this study we anticipate to explore proteome dynamics in the context of ASD and capture both differences in cellular composition of organoids and cell autonomous changes. Through our findings we hope to deepen our understanding of the molecular underpinnings of this intricate disorder and pave the way for the development of more precise diagnostic methods and targeted therapeutic interventions.

G 09
Investigating the role of Arabidopsis HISTONE DEACETYLASE 14 in chloroplasts

Presenting author:

University Münster, IBBP AG Finkemeier, Schlossplatz 7, 48149 Münster [DE], florian.kotnik@uni-muenster.de

Author(s):
Florian Kotnik, Claudia Markiton, Jürgen Eirich, Iris Finkemeier

Lysine acetylation is an important post-translational protein modification that plays a vital role in plant development and in responses to different environmental stimuli. Histone deacetylases (HDACs) are responsible for removing lysine acetylation on various proteins. While most work has focussed on the role of Arabidopsis HDACs on histone acetylation, their role in the deacetylation of non-histone proteins is much less known, although proteins of many different organelles have been found to be lysine-acetylated. From the 18 HDACs found in Arabidopsis, only HDA14 has been found to be dual-localized in plastids and mitochondria. Here we performed a quantitative mass spectrometry-based approach, using isobaric TMT labelling, to profile the lysine acetylome of an Arabidopsis hda14 mutant compared to WT. We identified 1509 acetylation sites on 881 Arabidopsis protein groups, of which 56 sites were de-regulated in the hda14 mutant. Most of these sites were derived from chloroplast proteins. In addition, we used different co-immunoprecipitation approaches to identify possible interaction partners of HDA14 and to identify its function in the regulation of organellar metabolism.

M 03
Edman degradation relaunched for unequivocal analysis of disulfide-rich peptides

Presenting author:

University of Bonn , Pharmaceutical Biochemistry and Bioanalytics, An der Immenburg 4, 53121 Bonn [DE], tkuhl@uni-bonn.de

Author(s):
Yomnah Y. Elsayed, Karl G. Wagner, Toni Kühl, Diana Imhof

N-terminal sequencing introduced by Peer Edman in 1949, was the gold standard for protein analysis for many years til the 1990s, when the rise of mass spectrometry superseded the stepwise chemical degradation of proteins in this field til today [1,2]. However, several scientific problems cannot be easily solved without the application of Edman degradation, e.g., the differentiation of isoleucin and leucin in protein sequences, the analysis of immobilized compounds or the identification of posttranslational modifications [3,4]. The analysis of disulfide bonds in cysteine-rich peptides is another tempting application for N-terminal sequencing. However, this requires the (re)establishment of suitable standard compounds and the development of a protocol for the rapid analysis of the disulfide connectivities in peptide and protein sequences. We present the application of such standard compounds in an optimized workflow in which partial reduction, alkylation, fractionation and Edman degradation are applied in a sequential manner. Peptides of different complexity (different length, 1-3 disulfide bridges), such as the conotoxins CCAP-vil and µ-KIIIA, were analyzed. With this study, we aim to relaunch N-terminal sequencing by applying and developing favorable protocols for rapid analysis exploiting this method. [1]Edman (1949) Arch Biochem 22,475 [2]Stehen, Mann (2004) Nat Rev Mol Cell Biol 5,699-711 [3]Lukas et al. (2022) Biol Chem 403,1099-1105 [4]Fitzner et al. (2023) Food Chem 136698

G 05
Establishing a cytosolic version of the mitochondrial processing peptidase to study mitochondrial protein import

Presenting author:

RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], lenhard@rhrk.uni-kl.de

Author(s):
Svenja Lenhard

Mitochondria consist of many hundreds of different proteins that are synthesized on cytosolic ribosomes. Mitochondrial protein import mechanisms have been extensively studied in the past. Aminoterminal presequences ensure the reliable targeting of client proteins into mitochondria. Subsequently to the import of these proteins, the presequences are proteolytically removed in the mitochondrial matrix by the mitochondrial processing peptidase, MPP. Strikingly, the processes occurring right before the translocation of a polypeptide remain unclear. In order to better understand the timing of the synthesis and import of precursor proteins, we engineered a yeast strain which expresses MPP in the cytosol. Expression of this cytosolic MPP (cytoMPP) is highly toxic as MPP cleavage in the cytosol obviously competes with mitochondrial import of precursor proteins. Establishment of this tool is expected to provide novel insights into (1) how different precursors are sequestered to the mitochondrial surface, (2) the determinants of post- or co-translational protein import, (3) which proteins are particularly sensitive to cleavage by cytoMPP and (4) which factors determine the import efficiency into mitochondria. Furthermore, we aim to characterize the conserved C-terminus of the MPP α-subunit concerning thus far unknown structure-function relationships. Therefore, this study aims to investigate both, endogenous as well as cytosolic MPP.

G 29
The natural small molecule compound prodigiosin targets the Golgi stacking protein GRASP55/GORASP2

Presenting author:

Heinrich Heine Universität, MPL / BMFZ, Universitätsstraße 1, 40225 Düsseldorf [DE], thomas.lenz@hhu.de

Author(s):
Thomas Lenz, Lena Berning, Ann Kathrin Bergmann, Björn Stork, Kai Stühler

Background Prodigiosin is a bacterial secondary metabolite that has been shown to have anticancer, antimalarial, antibacterial and immunomodulatory properties. It has been reported to affect cancer cells but not non-malignant cells, making it a promising lead compound for anticancer drug discovery. A direct protein target has not yet been experimentally identified. Methods In order to identify target proteins of prodigiosin, mass spectrometry-based thermal proteome profiling was used in its temperature range (TPP-TR) and compound concentration range (TPP-CCR) variants. TPP-TR was performed such that effects of prodigiosin treatment on both protein thermal stability and protein abundance could be determined simultaneously using the ratio-based thermal shift assay analysis (RTSA). Target validation was performed by a genetic knockout approach and electron microscopy. Results The Golgi stacking protein GRASP55/GORASP2 was identified as a target protein of prodigiosin. Among the prodigiosin-affected proteins (TPP-TR/RTSA), GRASP55 was the statistically most significant thermally stabilized protein with the lowest EC₅₀ (2.6 nM, TPP-CCR). Prodigiosin treatment severely affects Golgi morphology and functionality, and prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. Furthermore, prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux probably involving also other mechanisms such as organelle alkalization.

S 01
Bridging top-down proteomics and native mass spectrometry: A consortium-based study

Presenting author:

Technical University of Darmstadt, , Peter-Grünberg-Strasse 4, 64287 Darmstadt [DE], frederik.lermyte@tu-darmstadt.de

Author(s):
Frederik Lermyte

Native mass spectrometry allows the study of the quaternary structure of protein complexes, while top-down protein analysis provides proteoform-specific insights into the structure of individual protein chains. The combination of both methods – i.e., top-down fragmentation after native ionisation – allows the study of how specific proteoforms interact to form complexes. This powerful combination has led to important biological insights in recent years; however, due to a lack of standardisation, only a handful of labs regularly carry out this type of work. Here, we have brought together an international consortium of users with different experience levels, and have developed and tested standard protocols for native MS combined with top-down fragmentation. QTOF, Orbitrap, and FTICR instruments were all represented. The set of samples contained monomeric proteins as well as complexes, and water-soluble as well as membrane proteins. All participants successfully ionised and activated at least part of the set of native-like proteins, resulting in monomer ejection and backbone fragmentation. Both native precursor spectra and fragmentation patterns were remarkably consistent between labs. Overall, this work provides an entry point for newcomers to combine native with top-down MS. It serves as a robust benchmark for the expected results of such an experiment, and shows that these results are more dependent on inherent properties of the protein than on precise experimental conditions.

M 02
Nano-flow HILIC-MS-based site-specific assessment of RNA modifications

Presenting author:

Goethe University, Faculty of Biochemistry, Chemistry, Pharmacy, Max-von-Laue-Str. 9, 60439 Frankfurt am Main [DE], li@pharmchem.uni-frankfurt.de

Author(s):
Chengkang Li, Stefanie Kaiser

RNAs might undergo multiple modifications (epitranscriptome) post-transcriptionally, affecting their structures and functions accordingly, some of which may involve in disease development, e.g. cancers. Therefore, a better understanding of the modification type, quantity, and location in RNA will be beneficial to the mechanism study of disease and the development of targeted therapeutic drugs. Accurate identification and quantification of multiple RNA modifications are recently achieved using advanced mass spectrometric approaches, e.g. the Nucleic Acid Isotope Labeling Mass Spectrometry (NAIL-MS). However, a robust approach for site-specific localization of RNA modifications is still an unsolved but promising challenge in epitranscriptomic study. In order to avoid potential practical limitations, e.g. impaired MS sensitivity and instrument contamination, brought by the most popular ion-pairing reagent assisted reverse phase chromatography in LC-MS-based epitranscriptomic study yet, we separate different lengths (up to 30 base pairs long) of oligonucleotides using a cleaner ion-pairing reagent free technique, i.e. hydrophilic interaction chromatography, particularly under nano-flow. In the following MS analyses, a nearly infinite signal-to-noise ratio is recorded in the corresponding (MS1) extracted ion chromatogram by only injecting samples in “ng” magnitude, along with great (≥ 73%) MS2 fragmentation coverage rates under data-dependent acquisition mode.

G 08
Secretome Analysis Revealing Effects of Kallikrein-related Peptidase 6 (KLK6) in Pancreatic Ductal Adenocarcinoma

Presenting author:

Uniklinik Freiburg, Institute of Clinical Pathology, Breisacher Strasse 115a, 79106 Freiburg im Breisgau [DE], mujia.li@uniklinik-freiburg.de

Author(s):
Mujia Li, Bettina Wehrle, Patrick Bernhard, Janina Werner, Oliver Schilling

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease involved in inflammatory pathways. Additionally, its overexpression was detected in several tumor entities including PDAC. However, distinct substrates of KLK6 are only sparsely identified and the extent of its biological effects remains to be fully understood. This study aims to elucidate biological mechanisms of KLK6 in a MiaPaCa2 knockdown model. Cell conditioned medium was used for explorative proteomic analyses, revealing over 1400 secreted proteins. By using an isobaric labeling approach (TMT 16-plex), a quantitative analysis and comparison between control and KLK6 knockdown condition was achieved. Differential abundances of KLK6-related proteins suggest that KLK6 is part of a homeostatic system with feedback controls to maintain its equilibrium. Furthermore, effectors of the extracellular matrix were significantly differentially regulated, proposing an impact of KLK6 on extracellular matrix remodeling. As expected, we detected several pro- and anti-inflammatory proteins (PTX3, TGFB2, CFH, PTGDS, CCL6, IL1R1, S100A16) differentially regulated in the knockdown condition compared to control. Altogether, this study represents first secretome analysis to unravel the biological effects of KLK6 in PDAC. Given that KLK6 is already considered as a therapeutic target, our findings promise to furnish crucial and valuable insights into the physiological mechanisms that could be influenced by KLK6 inhibition.

G 21
Proteomics-based evaluation of different cell culture models for the development of treatments for psoriasis

Presenting author:

PharmBioTec GmbH, Drug Delivery, Am Nusskopf 32, 66578 Schiffweiler [DE], s.lichtner@pharmbiotec.de

Author(s):
Simone Lichtner, Kathrin Schunck, Carina Groh, Marc Schneider, Marius Hittinger

As a replacement for animal testing, novel cell culture models offer a promising opportunity for the assessment of safety and efficacy of drugs. Of particular interest in this context are co-culture models, which consist of several cell types and thus can make a precise prediction for human-relevant data. However, these models have often not been fully characterized yet. Proteomic profiling of different cell culture models can help to elucidate the functional interaction and lead to an optimization of the models. This knowledge will then be used to test different drugs for psoriasis. Finally, the mode of action of the active ingredients within different cell culture models will be investigated.

M 06
Assessment of cellular redox regulation via proteomics: Establishment of an appropriate sulfenic acid labeling procedure in human bronchial epithelial cells

Presenting author:

Karlsruhe Insitute of Technology (KIT), Food Chemistry and Toxicology, Adenauerring 20a, 76131 Karlsruhe [DE], martin.link@kit.edu

Author(s):
Martin Link, Jana Kuhn, Marlene Parsdorfer, Andrea Hartwig

Cysteine sulfenic acids occur as short-lived intermediates, and their detection appears to be a sensitive indicator of redox-regulated pathways. By using a sensitive labeling approach, we aim to identify proteins being redox-regulated by oxidative stress and to investigate the effects of toxic metal compounds on cellular redox regulation. Based on the approach published by Alcock et al. (Chembiochem 2020, 21, 1329–1334), we applied sulfenic acid labeling using the norbornene-biotin (norb-bio) probe in human bronchial epithelial cells. To define appropriate treatment conditions for reliable protein identification by LC-MS/MS, cells were first incubated with norb-bio, followed by oxidative stimulation with H₂O₂. With respect to sulfenic acid detection by streptavidin-HRP, BEAS-2B cells showed a dose-dependent increase in sulfenic acid formation after H₂O₂ stimulation. We found that 1.5 mM norb-bio for 2 h and an H₂O₂ stimulation with 150 to 200 µM for 1°h was sufficient for the labeling in intact BEAS-2B cells. However, since only very low levels of biotinylated protein were obtained, an efficient LC-MS/MS sample processing method needs to be selected. Up to now, we demonstrated that protein sulfenic acid is formed in BEAS-2B cells upon oxidative stimulation. Next, the LC-MS/MS measurement conditions will be adjusted in order to obtain an optimal readout to identify specific proteins involved in redox regulation, as well as the impact of toxic metal ions on this process.

G 26
Biogenesis of the presynaptic compartment

Presenting author:

Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Molecular Physiology & Cell Biology, Robert-Roessle-Strasse 10, 13125 Berlin [DE], lucht@fmp-berlin.de

Author(s):
Max Thomas Lucht, Filiz Sila Rizalar, Dmytro Puchkov, Volker Haucke

One of the most striking properties of a neuron is the great distance between its soma and the presynapses (up to one meter in humans). Since the majority of proteins are synthesized in the soma, the delivery of the machinery necessary to form functional synapses poses a unique challenge. To improve the understanding of this transport process, how these precursor vesicles (PV) might be assembled and their transport regulated, it is crucial to investigate their fundamental properties. To this end, uncovering their protein composition and their ultrastructure will help to integrate the transport of presynaptic proteins into the overarching neuronal processes. The main challenges arise from the transient nature of PVs as well as their large proteomic overlap with the vastly more abundant synaptic vesicles. In this project we investigate the protein composition of PVs as well as their structural identity. By redirecting PVs to mitochondria, an electron-dense organelle, and combining conventional live imaging with focused ion beam scanning electron microscopy (FIB-SEM), we are able to show their high morphological variability. To study their protein composition via mass spectrometry, especially in terms of sorting factors and regulators, it will be necessary to isolate a large number of vesicles with a sufficient purity. Therefore, we will pursue multiple strategies that target PV cargo proteins as well as the main motor protein responsible for PV transport.

Sept 5, 13:15
Advances in Mass spectrometry-based proteomics for body fluid and single cell type-resolved tissue proteomics

Presenting author:

Max Planck Institute of Biochemistry, Martinsried, Germany

Author(s):
Matthias Mann

Recent breakthroughs in high-content imaging, mass spectrometry-based proteomics and computational biology are transforming bioscience. In this talk, I will introduce our Python-based open-source AlphaPept software suite, designed for rapid and efficient processing of large MS datasets. Additionally, I will highlight our advancements in MS-based technologies, enable large scale interactomics studies as well as large plasma-cohort analysis to identify diagnostic and prognostic biomarkers of chronic diseases. Finally, I will describe our new workflow termed Deep Visual Proteomics that enables single cell analysis to describe cellular heterogeneity, such as those that arise in cancer. DVP combines high-content microscopy, AI-driven image recognition, and laser microdissection with ultrahigh sensitivity MS to connect visual, spatial, and molecular proteomics data. Applied to various diseases, such as borderline ovarian cancers, rare cutaneous drug reactions, and liver diseases, this provides a comprehensive understanding of cellular function at resolution specific to the cell type, identifying potential therapeutic targets. DVP has great potential in facilitating diagnosis and prognosis and moving us towards personalized cancer medicine, which is our ambition moving forward.

Short talk 6
µPhos: a scalable and sensitive platform for functional phosphoproteomics

Presenting author:

Universitätsklinikum Jena, Funktionelle Proteomanalyse, Am Klinikum 1, 07747 Jena [DE], florian.meier@med.uni-jena.de

Author(s):
Denys Oliinyk, Andreas Will, Felix Schneidmadel, Maximilian Böhme, Jenny Rinke, Andreas Hochhaus, Thomas Ernst, Markus Lubeck, Oliver Raether, Sean Humphrey, Florian Meier

Mass spectrometry has revolutionized cell signaling research by vastly simplifying the analysis of protein phosphorylation on a systems scale. However, disentangling the functionality of the phosphoproteome remains a particularly challenging task, considering that only few of the well over 100,000 reported phosphorylation sites have known cognate kinases, and even fewer are functionally characterized. There is therefore a growing need to further increase the throughput, sensitivity and robustness of MS-based phosphoproteomics workflows to study cellular responses to perturbations in space and time. Here we introduce µPhos (‘microPhos’), an accessible phosphoproteomics platform that permits phosphopeptide enrichment from 96-well cell culture experiments in 30,000 unique phosphopeptides in a human cancer cell line using 20 µg starting material, and confidently localize ~6,500 phosphosites from 1 µg. This depth covers key signaling pathways, rendering sample-limited applications and perturbation experiments with hundreds of samples viable as we demonstrate by profiling the time-resolved response of a chronic myeoloid leukemia model to tyrosine kinase inhibitors.

G 28
Detection of known and novel small proteins in Pseudomonas stutzeri using a combination of bottom-up and digest-free proteomics and proteogenomics

Presenting author:

MPI of Biophysics, , Max-von-Laue-Str. 3, 60438 Frankfurt [DE], jakob.meier-credo@biophys.mpg.de

Author(s):
Jakob Meier-Credo, Benjamin Heiniger, Christian Ahrens, Julian Langer

Small proteins of around 50 aa have been largely overlooked in biochemical assays due to the inherent challenges with detecting and characterizing them. Recent discoveries of their critical roles in many biological processes have led to an increased recognition of the importance of small proteins for basic research and as potential new drug targets. One example is CcoM, a 36 aa subunit, that plays an essential role in adaptation to oxygen-limited conditions in P.stutzeri, a model for the clinically relevant pathogen P. aeruginosa. However, as no comprehensive data were available in P. stutzeri, we devised an integrated, generic approach to study small proteins more systematically. Using the first complete genome as basis, we conducted proteomics analyses and established a digest-free, direct-sequencing approach to study cells grown under aerobic and oxygen-limiting conditions. Finally, we also applied a proteogenomics pipeline to identify missed protein-coding genes. We identified 2921 known and 29 novel proteins, many of which were differentially regulated. Among 176 small proteins 16 were novel. Direct sequencing exhibited advantages in the detection of small proteins with higher sequence coverage and more PSMs. Three novel small proteins, uniquely identified by direct sequencing and not conserved beyond P. stutzeri, were predicted to form an operon with a conserved protein and may represent de novo genes.

M 08
APEX-based proximity labeling for time-resolved, subcellular proteomics of primary cilia to study proteome dynamics during active signaling

Presenting author:

Saarland University Medical Center, Medical Biochemistry and Molecular Biology, Kirrberger Str. 100, 66421 Homburg [DE], david.mick@uks.eu

Author(s):
Tommy Sroka, Elena May, David Mick

The primary cilium is a fL-sized compartment of vertebrate cells that initiates signaling cascades in response to external stimuli. Effective cilia signaling depends on the dynamic transport of signaling components such as receptors and effectors into and out of the cilium. Yet, the precise content and the extent of the proteomic remodeling of primary cilia during active signaling remained largely unknown. We employ proximity labeling methods using cilia-localized ascorbate peroxidase (cilia-APEX) in combination with tandem-mass-tags for quantitation by synchronous precursor selection-MS³ approaches to profile the cilia proteome in a time-resolved manner after signal pathway stimulation. By inducing the hallmark primary cilium signaling pathway, Hedgehog signaling, we could reconcile known dynamics in the localization of known signaling components. We further revealed a fast removal of the cAMP-dependent protein kinase (PKA) holoenzyme, including its unconventional A-kinase anchoring protein GPR161 from primary cilia. Hierarchical clustering identified the putative phosphatase PALD1 that accumulates in cilia in response to active Hedgehog signaling to dampen signaling in a cell type-specific manner. Our unbiased analyses demonstrate that proximity labeling in combination with quantitative proteomics allows time-resolved proteomics of subcellular compartments and provide novel insights into how primary cilia orchestrate signaling processes.

G 16
Identification of mitotic DNA-protein complexes formed after replication stress by ChIP-MS analysis

Presenting author:

Kaiserslautern University, , Fruchthallstraße, 67655 Kaiserslautern [DE], fmohseni@rptu.de

Author(s):
Farbod Mohseni, Angela Wieland, Andrea Tirincsi, Markus Räschle

Replication stress challenges genome stability, leading to replication fork stalling or collapse. Incomplete replication gives rise to mitotic errors, including mis-segregation of chromosomes and formation of ultrafine bridges (UFBs) connecting segregating sister chromatids. Most UFBs disappear during late mitosis, however the mechanism of UFB resolution remains unknown. In this study, we apply Chromatin Immunoprecipitation Mass Spectrometry (ChIP-MS), a powerful technique widely used for the characterization of protein-DNA complexes formed during transcription or DNA repair. ChIP-MS involves crosslinking of transient protein assemblies with the bound DNA, isolation of chromatin and its fragmentation. Solubilized protein-DNA complexes are then subjected to immune precipitation and quantitative MS analysis. Here we conduct ChIP-MS experiments using antibodies recognizing known UFB-associated proteins, including the BLM and PICH helicase, as well as the DNA repair factor FancD2. By comparing the formation of protein assemblies induced by mild replication stress in S-phase and mitotic cells, we aim to identify novel UFB-associated proteins. Currently, we are in the process of validating various mitosis-specific interaction partners through reciprocal ChIP-MS experiments, immunofluorescence microscopy, and phenotypic assays. The identification of novel UFB-associated proteins will enhance our understanding of UFB resolution and their role in maintaining genomic stability.

Sept. 5, 16:00
Native mass spectrometry: How to probe molecular principles of assembly and interactions of protein complexes

Presenting author:

Frankfurt University, Physical and Theoretical Chemistry, Max-von-Laue-Str. 9 , 60438 Frankfurt [DE], morgner@chemie.uni-frankfurt.de

Author(s):
Nina Morgner

Protein complex assembly as well as their interplay are controlled by the non-covalent interactions of all biomolecular partners. Native mass spectrometry and ion mobility are ideally suited to unravel the molecular principles which tightly control these interactions. Here I will present what we can learn about well-choreographed assembly strategies of a multi protein complex such as an ATPase or rather unwanted aggregation as seen for the Alzheimer related Amyloid b peptide. For the example of photoreceptors I will show how instrumental modifications can allow for time resolved studies of light dependent conformational rearrangements upon illumination.

G 10
Single cell proteome analysis with ultra-high sensitivity using a timsTOF mass spectrometer

Presenting author:

Bruker Daltonics GmbH + Co KG, , Fahrenheitstr 4, 28359 Bremen [DE], tor.mueller@bruker.com

Author(s):
Christoph Krisp, Anjali Seth, David Hartlmayr, Torsten Müller, Guilhem Tourniaire, Markus Lubeck, Gary Kruppa

For single cell proteome analysis, ultra-high sensitivity mass spectrometry is a key to reach proteome coverages necessary for understanding the cellular heterogeneity on a cell-by-cell level. Latest enhancements in ion transfer with a larger transfer capillary, an additional higher-pressure segment for more effective ion collection and two orthogonal deflections, to maintain robustness, and high-capacity trapped ion mobility spectrometry (TIMS) pushes the limits of detection to single cell level. Here, we assessed the sensitivity of a timsTOF Ultra mass spectrometer using a dilution series of K562 cell digest showing excellent identification rates, reproducibility, and quantification accuracy per concentration replicates. Processing of the dia-PASEF data identified >1,000 protein groups out of 15 pg, and >7,000 protein groups out of 16 ng K562 peptides loaded on column. The quantitative accuracy improved inversely with loaded peptide amounts with 19% at 15 pg to 4% at loads of 4, 8 and 16 ng. Analysis of the isolated HeLa cells resulted in good identification rates and good reproducibility per individual cell count group with expected increase in protein abundance from the single cells to 20 cells. The timsTOF Ultra combined with automated single cell isolation and sample preparation using the cellenONE® platform for protein-loss reduced preparation and transfer with the proteoCHIP format leads to deep proteome coverage and high reproducibility.

Sept. 5, 16:30
Narrow-window DIA for ultra-fast quantitative analysis of comprehensive proteomes with high sequencing depth

Presenting author:

University of Copenhagen, Novo Nordisk Foundation Center for Protein Research, Blegdamsvej 3b, 2200 Copenhagen [DK], jesper.olsen@cpr.ku.dk

Author(s):
Jesper Olsen

The goal of mass spectrometry (MS)-based proteomics is to efficiently and reliably characterize complete proteomes. We introduce a narrow window data-independent acquisition (DIA) method utilizing 2-Th precursor isolation windows. This method dissolve the distinctions between data-dependent acquisition (DDA) and DIA approaches. To achieve this, we make use of the new Quadrupole Orbitrap mass spectrometer coupled to an asymmetric track lossless (Astral) analyzer, which offers exceptional features such as >200 Hz MS/MS scanning speed, high resolving power, high sensitivity, and low ppm-mass accuracy. By using narrow-window DIA, we are able to profile >100 full yeast proteomes within a single day or approximately 10,000 human proteins in just half-an-hour. Additionally, by acquiring multiple shots of fractionated samples, we can comprehensively cover human proteomes in approximately 3 hours. This approach demonstrates a similar level of depth as next-generation RNA sequencing, but with 10 time’s higher throughput compared to the current leading MS techniques. We demonstrate high quantitative precision and accuracy based on 3-species mixture analysis. Overall, our ultra-fast scanning narrow window DIA strategy offers a significant advancement in proteomics research, enabling rapid and accurate protein characterization with impressive throughput and quantification capabilities.

M 01
Identification of novel cellular targets of α-13’-COOH and garcinoic acid using a compound-centric chemoproteomic method

Presenting author:

Friedrich Schiller Universität Jena, Institute of Nutrition, Dornburgerstrasse 25, 07743 Jena [DE], sylvia.omage@uni-jena.de

Author(s):
Sylvia Omage, Maria Wallert, Stefan Lorkowski

Identifying the cellular targets of novel natural products increases the understanding of their biological actions. Most proteomic approaches lead to numerous false-negative/false-positive hits. We present an optimised chemoproteomic method that leads to a streamlined list of targets of α-13’-COOH and garcinoic acid (GA). Our approach is particularly suitable for hydrophobic compounds, since the methacrylic resin used is resistant to organic solvents and extreme pH, unlike the more commonly used sepharose resin. With the optimized approach α-13’-COOH and GA were separately coupled to the insoluble methacrylate resin, Toyopearl AF amino 650M, using their carboxyl groups. The coupled resins were incubated with appropriately processed cell lysates. After extensive washing of the resin, the proteins bound to the resin-coupled compounds were eluted and identified using mass spectrometry. We found 17 proteins involved in lipid metabolism, antioxidant response, glucose metabolism as well as stress and immune response. We have validated one of the targets, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), using western blotting. Since HMGCS1 is involved in cholesterol synthesis, this target is in line with previous findings that α-13’-COOH regulates cellular lipid metabolism. Validation of the remaining proposed targets is ongoing. In conclusion, compound-centric chemoproteomics has enabled us to identify HMGCS1 as a potential molecular target of α-13’-COOH and GA.

O 03
Coordination of DNA damage and aging by ubiquitin signaling and the ubiquitin-proteasome system

Presenting author:

CECAD Research Center , , Joseph-Stelzmann-Straße, 26, 50931 Köln [DE], mpandrea@uni-koeln.de

Author(s):
Maria Pandrea, Thorsten Hoppe

Double strand breaks (DSBs) are severe types of DNA lesions that if left unrepaired can lead to genomic instability and premature ageing. We are using the C. elegans germline to study homologous recombination DSB repair and its regulation by ubiquitin signaling. It remains unclear whether the age-associated decline in ubiquitin proteasome system (UPS) efficiency contributes to age-related genomic instability and disease progression. The E3/E4 ubiquitin ligase UFD-2 accumulates in the nuclei of irradiated germ cells within ubiquitination hubs to regulate DSB repair and DNA damage-induced apoptosis. Therefore, we are investigating the tissue-specific function of UFD-2 to characterize the coordination between DNA damage and aging by ubiquitin signaling and the UPS. Using an integrated proteomics approach consisting of large-scale proteomic studies and biotinylation-based protein-protein interaction assays we will generate a list of potential co-factors and substrates of UFD-2. So far, we have identified a putative UBQL4 ortholog F49C12.9 as an interactor of UFD-2 capable of regulating DSB responses. In addition, we observe that upon UFD-2 loss worms fail to inhibit RNA processing, ribosomal assembly and translation-associated processes following genotoxic insults. We expect to obtain an extensive picture of how genotoxic stress controls localization and activities of repair factors and how this is coordinated by ubiquitin signaling dynamics during development and aging.

C 04
Proteome analysis of precursor lesions from pancreatobiliary cancer to improve early cancer diagnostic

Presenting author:

Molecular Proteomics Laboratory, BMFZ, Heinrich Heine Universität, Düsseldorf, 22.07., Universitätsstr. 1 , 40225 Düsseldorf [DE], stella.pauls@hhu.de

Author(s):
Stella Pauls, Anja Stefanski, Christin Hafermann, Friederike Opitz, Sandra Biskup, Irene Esposito, Kai Stühler

Objective: Cell‑subpopulation analysis for the detection of proteins that are involved in tumor progression, enables the potential to find new disease-specific biomarker to improve diagnostics. Methods for morphomolecular characterization of pancreatobiliary (PB) precursors as well as molecular subtyping of different precursor stages applied on FFPE tissues has been combined with quantitative proteomics. Methods: For label-free analysis of pancreatic FFPE tissue, a modified tissue lysis protocol is used to disrupt cells, reverse the formalin fixation and to extract proteins. For protein purification and processing for MS analysis an optimized protocol for a single-pot solid-phase-enhanced sample preparation (SP3) method was applied. Furthermore, an optimized data independent acquisition method (DIA) was applied for LC-MS/MS analysis and data processing was performed using DIA-NN. Results: Using these optimized methods, we are able to analyze 2.5 mm² FFPE pancreatic tissues (approx. 8,600 cells) and to identify around 2,700 proteins per slice. We apply these optimized methods on precise morphological characterized and microdissected areas of intraductal papillary mucinous neoplasm (IPMN) from pancreatic FFPE tissues and identify over 5,000 proteins in 53 tissue slices of different tissue types. By using ANOVA and soft clustering methods, it is possible to find interesting biomarker candidates and obtain deeper insights of biological processes involved in tumor progression.

B 01
Exploring Pathogenic Mutations on Phosphorylation Sites: Unraveling Disease Mechanisms via Interactome Studies

Presenting author:

Max Delbrück Center for Molecular Medicine, Proteome Dynamics, Robert Rossle str. 10, 13125 Berlin [DE], trendelina.rrustemi@mdc-berlin.de

Author(s):
Trendelina Rrustemi

With the advancement of sequencing technologies, identification of single nucleotide mutations surged, exceeding functional characterization capacity. Many of these mutations occur within structure-lacking intrinsically disordered regions (IDRs) of proteins. IDRs often contain short linear motifs (SLiMs) that are crucial for protein-protein interactions (PPIs) and are often subject to phosphorylation. Our approach involved immobilizing synthetic peptides representing mutated IDR regions onto cellulose membranes to capture interacting proteins from cellular extracts. This enabled simultaneous comparison of interaction partners between wild-type, phosphorylated, and mutated peptide forms, allowing functional assessment of individual mutations. We screened 36 disease-causing phosphorylation site mutations within IDRs, sourced from PTMVar database. The results revealed substantial differences between phosphorylated and mutated peptide interactomes, often due to disrupted phosphorylated SLiMs. We later focused on S102P mutation in GATAD1 that is linked to dilated cardiomyopathy. We found that the mutation disrupted a phosphorylation site crucial for interaction with 14-3-3 proteins. Further studies suggest that the GATAD1 peptide is important for nuclear localization and 14-3-3 binding prevents importin-GATAD1 interaction, highlighting its importance in proper nucleocytoplasmic transport.

G 06
In-depth exploration of the cyanobacterial secretome with trapped ion mobility spectrometry coupled to dia-PASEF

Presenting author:

Friedlich Schiller University Jena, Institute for Inorganic and Analytical Chemistry, Lessingstr. 8, 07743 Jena [DE], david.russo@uni-jena.de

Author(s):
David A. Russo, Denys Oliinyk, Florian Meier, Julie A. Z. Zedler

Extracellular proteins are involved in a remarkable number of fundamental processes in cyanobacteria. Yet, there is limited knowledge regarding the identity and function of these secreted proteins. Here, we develop an approach which combines single-pot, solid-phase-enhanced sample preparation (SP3) with trapped ion mobility spectrometry (TIMS), coupled to parallel accumulation-serial fragmentation with DIA (dia-PASEF) to enable description of the cyanobacterial secretome with unprecedented depth. Application to cyanobacteria from three distinct habitats, Synechocystis sp. PCC 6803, Synechcoccus sp. PCC 11901 and Nostoc punctiforme PCC 73102, allowed the identification of up to 62% of all predicted secreted proteins. The approach was then extended to compare the Synechocystis sp. PCC 6803 wild-type secretome with that of a bloom-like aggregated state and a secretion-impaired mutant. We also demonstrate that the method can be miniaturized and adapted to a 96-well format for high-throughput secretome analysis. These findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across species from different environments. Our approach should be broadly applicable to bacterio- and phytoplankton, with the potential to open new avenues of investigation in microbial exoproteomics.

M 10
ENRICH-iST technology provides deeper coverage of the plasma proteome

Presenting author:

Bruker Daltonik GmbH & Co. KG, Bremen, Germany, AppDev - BLSMS, Fahrenheitstrsse 4, 28359 Bremen [DE], Andreas.Schmidt@bruker.com

Author(s):
Andreas Schmidt, Katrin Hartinger, Claudia Martelli, Zehan Hu, Katharina Limm, Sebastian Mueller, Xaver Wurzenberger, Nils A. Kulak

As a liquid biopsy, blood is easily available and very rich in information on personal health and wellness. Moreover, its steady contact with all tissues and quick turnover time allows for precise determination of disease progression or the effect of a treatment. As a derivative after removing the cellular fraction, plasma retains the valuable information in form of antibodies, protein released from tissue or cytokines. Due to its high dynamic range, it presents a challenging sample for proteomics analysis. We introduce a fast and robust method to reduce the dynamic range in protein abundance by binding proteins to the micro-particles and follow up proteomics analysis of the bound fraction. In comparison to selective technologies, high abundant plasma proteins are still present in the sample. The ENRICH-iST technology, preserves quantitative differences in plasma samples and is therefore suitable to study disease cohorts or treatment progression with high reproducibility. A model cohort of plasma samples derived from lung cancer patients and matched healthy donors was prepared with the ENRICH-iST kit and analyzed by dia-PASEF technology on the TimsTOF HT mass spectrometer. Applying a sample derived spectral library, we were able to cover more than 1500 proteins in both cohorts with only 30 min acquisition time/sample. Using the ENRICH technology, tripled the number the number of significantly enriched proteins, thus allowing for a more precise description of the disease state.

G 23
Characterizing mitochondrial protein import in senescence

Presenting author:

Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], jo.schmidt@med.uni-frankfurt.de

Author(s):
Jonas Schmidt, Christian Münch

During aging, senescent cell accumulation occurs, characterized by irreversible cell-cycle arrest, pro-inflammatory phenotype, and decline in proteostasis. Senescence also leads to changes in mitochondria, including enlargement, increased mass, reduced ATP production, and decreased membrane potential. These alterations likely affect mitochondrial protein import (MPI) that relies on membrane potential and ATP. Consequently, MPI impairment is highly plausible in aged mitochondria. Restoring membrane potential has been shown to extend the lifespan in C. elegans, and genetic variations in the mitochondrial protein import system are linked to lifespan differences in humans. Despite the significance of MPI, the impact of senescence on this process is largely unknown. This project aims to address this knowledge gap using mePRODmt, a SILAC-based proteomics approach for quantifying protein uptake into mitochondria in IMR90 cells. We will investigate alterations in MPI during senescence and examine affected pathways in the senescent phenotype. Understanding the impact of senescence on mitochondrial protein import is crucial for unraveling the complex interplay between aging, cellular homeostasis, and neurodegenerative diseases.

M 13
TurboID reveals the proxiomes of VIPP1 and VIPP2 in Chlamydomonas reinhardtii and confirms VPL2 and PGRL1 in the VIPP1 proxiome

Presenting author:

RPTU Kaiserslautern-Landau, Molecular Biotechnology & Systems Biology, Paul-Ehrlich-Str 23, 67663 Kaiserslautern [DE], m.schroda@rptu.de

Author(s):
Elena Kreis, Katharina König, Melissa Misir, Justus Niemeyer, Frederik Sommer, Michael Schroda

In Chlamydomonas reinhardtii, VIPP1 and VIPP2 play roles in the sensing, signaling and coping with membrane stress, triggering a chloroplast unfolded protein response (cpUPR), and in the biogenesis of thylakoid membranes. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. TurboID-mediated PL with VIPP1/2 as baits under ambient and H₂O₂ stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B and CDJ2. Novel proteins in the VIPP1/2 proxiome can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport. A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL1-11). We confirmed VIPP1 in the proxiomes of VPL2 and PGRL1 in reciprocal experiments and aim to (co)-localize them in the chloroplast. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs and their proximal proteins in thylakoid biogenesis and stress responses.

C 03
Proteomic Characterization of Colorectal Cancer Patients for Precision Oncology

Presenting author:

German Cancer Research Center (DKFZ), Molecular Genome Analysis, Im Neuenheimer Feld 280, 69120 Heidelberg [DE], luisa.schwarzmueller@dkfz-heidelberg.de

Author(s):
Luisa Schwarzmüller, Efstathios Vlachavas, Katja Beck, Katrin Pfütze, Theresa Mullholland, Johannes Betge, Stefan Fröhling, Dominic Helm, Stefan Wiemann

Although research has made major advances in the discovery of cancer biomarkers and the development of new therapeutic options, the majority of patients receive the standard treatment for their respective cancer type. Molecular tumor boards, such as within the NCT MASTER program, try to leverage recent technological developments for in-depth molecular tumor characterization to infer personalized therapy recommendations. This genome-driven precision oncology project considers each patient’s mutational status and mRNA expression for treatment guidance. However, including protein abundance and phosphorylation status would offer an additional layer of tumor characterization regarding cancer pathway activities. To advance the integration of high-throughput, unbiased proteomics into precision oncology, we established a mass spectrometry-based full and phospho proteome screening of tissue samples and applied it to a retrospective NCT MASTER cohort of 31 colorectal cancer patients. Adding the informational layers of protein expression and activity to the previously acquired genomic and transcriptomic information, offered new insights into oncogenic mechanisms and identified possible tumor vulnerabilities. The protein and pathway activity measurements could have a significant impact on improving the stratification of patients into more actionable treatment “baskets” and enhance personalized oncology.

Sept 6, 11:45
An Integrated Landscape of mRNA and Protein Isoforms

Presenting author:

Max Delbrück Center, Berlin, Germany

Author(s):
Matthias Selbach

Proteomic characterization of protein isoforms poses a significant challenge due to limitations in available methodologies. Current bottom-up proteomic approaches provide limited information on protein isoforms, while top-down proteomic workflows often fail to comprehensively capture them. In this study, we introduce peptide correlation profiling (PepCP) as a novel method for globally characterizing protein isoforms. PepCP involves protein fractionation via SDS-PAGE, followed by bottom-up proteomic analysis of individual fractions. By quantifying peptide abundances across protein fractions, we obtain peptide abundance profiles that enable identification of protein isoforms through a computational pipeline. Using PepCP, we identified approximately 20,000 protein isoforms for 10,000 genes in human RPE-1 cells. Our results demonstrate that PepCP can identify isoforms arising from diverse cellular mechanisms, such as alternative splicing, alternative translation, and proteolytic processing. Additionally, we complemented our proteomic data by conducting full-length mRNA sequencing. Our integrated landscape of mRNA and protein isoforms provides insights into how transcriptional, translational and post-translational processes contribute to proteome complexity.

O 02
A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae

Presenting author:

RPTU Kaiserslautern-Landau, Standort Kaiserslautern, AG Zellbiologie, Erwin-Schrödinger-Straße 13, 67663 Kaiserslautern [DE], simakin@rhrk.uni-kl.de

Author(s):
Pavel Simakin, Christian Koch, Johannes M. Herrmann

Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the baker’s yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.

M 07
Quantitative Translation and Import Proteomics using mePROD

Presenting author:

Institute of Biochemistry II, , Theodor-Stern-Kai 7, 60590 Frankfurt am Main [DE], tascher@med.uni-frankfurt.de

Author(s):
Georg Tascher, Jasmin Schäfer, Suleyman Bozkurt, Christian Münch

Measuring protein translation is an invaluable tool for understanding cellular stress-responses and protein homeostasis. Classic pulsed stable isotope labeling with amino acids in cell culture (pSILAC) requires relatively long pulse time for sufficient incorporation of heavy isotopes into the proteome. Hence, we developed multiplexed enhanced protein dynamics mass spectrometry (mePROD) combining pSILAC with Tandem mass tags (TMT), enabling robust quantification of translation on a proteome wide scale in experiments with short labeling times. This was achieved by incorporating a „booster-channel” containing only heavy-labeled peptides to increase acquisition of MS2-spectra and thus quantification of newly synthesized peptides as well as a “noise-channel“ containing only light peptides to determine background noise levels and co-isolation interference for each individual peptide. We recently expanded the method to study mitochondrial protein import by using a booster-channel comprised of enriched mitochondria. We show that mePROD provides an easy and cost-efficient method to profile proteome-wide translatome changes at a temporal resolution of minutes. The method already has brought valuable insight into different biomedical contexts, such as SARS-CoV2-Infection and acute myeloid leukemia. Notably, the noise-channel included in mePROD makes ratio compression, caused by co-isolation of non-targeted ions, as typically observed in TMT MS2-based methods, largely negligible.

G 19
Structural & Functional Analysis of MICOS & the Mitochondrial Intermembrane Space Bridging Complex (MIB)

Presenting author:

Universität des Saarlandes, Medical Biochemistry & Molecular Biology, Kirrberger Straße 100, Gebäude 45.2, 66421 Homburg [DE], martin.van-der-laan@uks.eu

Author(s):
Alexander von der Malsburg, Martin van der Laan

Mitochondria are surrounded by two distinct membrane systems. The outer membrane (OM) mediates communication with the cytosol and other organelles. The inner membrane (IM) is particularly protein-rich and harbors the machinery for ATP synthesis by oxidative phosphorylation. Intimate cooperation of both membranes is required for key functions of mitochondria, like lipid synthesis, channeling of metabolites an ions, like Calcium, and apoptosis. We and others have identified and initially described a direct OM-IM contact site in yeast mitochondria formed by the Mitochondrial Contact Site and Cristae Organizing System (MICOS) in the IM and the Sorting and Assembly Machinery (SAM) in the OM. Our recent proteomic and biochemical studies on this Mitochondrial Intermembrane Space Bridging (MIB) super-complex in human mitochondria have revealed a novel mechanism for the biogenesis of OM beta-barrel proteins, like VDACs, that requires the Hsp40 co-chaperone DNAJC11 at the MIB.

G 30 & Short talk 2
The proteomic landscape of synaptic diversity across brain regions and cell types

Presenting author:

MPI Brain Research, , Max-von-Laue Strasse 4, 60438 Frankfurt am Main [DE], marc.van-oostrum@brain.mpg.de

Author(s):
Marc van Oostrum, Thomas Blok, Stefano L. Giandomenico, Susanne tom Dieck, Georgi Tushev, Nicole Fürst, Julian Langer, Erin M. Schuman

Neurons diversify synaptic contacts using protein combinations that define the specificity and function of synapses. While there is ample evidence of diverse synaptic structures, states or functional properties, the diversity of the underlying individual synaptic proteomes remains largely unexplored. We used 7 different Cre-driver mouse lines crossed with a floxed mouse line in which the presynaptic terminals were fluorescently labeled (SypTOM) to identify the proteomes that underlie synaptic diversity. We used fluorescent-activated synaptosome sorting to isolate and analyze using quantitative mass spectrometry 18 types of synapses and their underlying synaptic proteomes. We discovered ~1’800 unique synapse type-enriched proteins and allocated thousands of proteins to different types of synapses. We identify commonly shared synaptic protein modules and highlight the hotspots for proteome specialization. A protein-protein correlation network classifies proteins into modules and their association with synaptic traits reveals synaptic protein communities that correlate with neurotransmitter identity. We reveal specializations and commonalities of the striatal dopaminergic proteome and highlighting proteome signatures that relate to the functional properties of interneuron synapse types. This study opens the door for molecular systems-biology analysis of synapses and provides a framework to integrate type-specific proteomic information with cellular or circuit-level experiments.

G 01
The electrophilic immunometabolite itaconate causes an acid stress response as well as S-bacillithiolation and S-itaconation in the thiol proteome of Staphylococcus aureus

Presenting author:

Freie Universitat Berlin, Institut für Biologie-Mikrobiologie, Königin-Luise-Straße 12-16, 14195 Berlin [DE], vu.v.loi@fu-berlin.de

Author(s):
Van Loi Vu, Tobias Busche, Susanne Eva Müller, Benno Kuropka, Karen Methling, Michael Lalk, Jörn Kalinowski, Haike Antelmann

Using RNA-seq transcriptomics and Northern blot transcriptional analyses, we analysed the specific stress responses caused by itaconate. Shotgun proteomics was applied to identify the targets of itaconation and S-bacillithiolation by itaconate in S. aureus. Phenotype analyses of mutants were used to analyse the role of specific defense mechanisms against itaconate stress. In the RNA-seq transcriptome, itaconate caused predominantly an acid stress response as revealed by the induction of the GlnR, KdpDE, CidR, SigB and GraRS regulons and the urease-encoding operon in S. aureus. The urease and urea supplementation were found to protect S. aureus from itaconate-induced acid stress. The generation of ROS and oxidative protein damage by itaconate was indicated by the up-regulation of the PerR, CtsR and HrcA regulons. Using shotgun proteomics, itaconate was shown to cause widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting the oxidative and electrophilic mode of action of itaconate in S. aureus. In phenotype analyses, the catalase KatA and the low molecular weight thiol bacillithiol (BSH) were found to provide protection against itaconate-induced ROS in S. aureus. Our results revealed that the antimicrobial mode of action of the itaconate in S. aureus is mediated by acid stress, oxidative and electrophilic stress, leading to S-bacillithiolation and itaconation

G 20
Unraveling the Link between Neuronal Activity Patterns and Proteome Remodeling through Optogenetic Stimulation and Mass Spectrometry Analysis

Presenting author:

Max Planck Institute for Brain Research, Synaptic Plasticity, Max von Laue Str. 4, 60438 Frankfurt [DE], quinn.waselenchuk@brain.mpg.de

Author(s):
Quinn Waselenchuk, Kristina Desch, Julian Langer, Erin Schuman

Understanding how neurons encode and process information is crucial for understanding synaptic transmission and plasticity. Indeed, neuronal activity patterns, represented by action potential firing, play a pivotal role in triggering downstream pathways and adaptive processes such as synaptic plasticity. Manipulating neuronal activity has been shown to induce changes in the transcriptome, proteome, and phosphoproteome, highlighting their interconnectedness. However, discrete temporal firing pattern-associated proteome dynamics remain unexplored. This project aims to fill this gap by tightly controlling and reading out neuronal activity using all-optical methods and determining neuronal proteomic and phosphoproteomic changes through mass spectrometry-based analysis. Primary cultured hippocampal neurons expressing light-gated ion channels will be subjected to defined firing patterns through light pulses, followed by collection of cells for (phospho)proteomic analysis. Proteomic changes occurring at synapses will be further assessed by comparing results from whole neurons with synaptosomal preparations. Additionally, ex vivo hippocampal slices will be isolated and stimulated, enabling assessment of response heterogeneity within the brain region. This comprehensive approach aims to uncover the relationship between neuronal activity patterns and their downstream proteomic and phosphoproteomic responses, shedding light on mechanisms underlying synaptic transmission and plasticity.

S 03 & Short talk 3
Insights into Meiosis: Elucidating DNA Repair Modulation via Mass Spectrometry

Presenting author:

Friedrich Miescher Laboratory, , Max-Planck-Ring 9, 72073 Tübingen [DE], john.weir@tuebingen.mpg.de

Author(s):
Veronika Altmannova, Petra Janning, Franziska Müller, Tanja Bange, John Weir

Exploring meiosis is key to understanding eukaryotic propagation and diversity. The pivotal process in meiosis I is accurate segregation of homologous chromosomes, facilitated by physical linkages - crossovers - derived from programmed double-strand DNA breaks. Crossovers are essential in the germline, yet deleterious in somatic cells, highlighting a unique DNA repair modulation in meiosis. Given the limited availability of mammalian germline tissue, our research utilizes budding yeast as a model system. Our work has been focused on the Mer3 helicase, known as HFM1 in mammals. Using immunoprecipitation coupled with mass spectrometry (IP-MS), we identified potential Mer3 interactors, including several DNA repair factors. We generated recombinant proteins and complexes and characterisded them using techniques including cross-linking mass spectrometry (XL-MS), which validated protein complex models produced by AlphaFold2. We hypothesised that phosphorylation might govern several protein complexes' formation. Hence, we studied the phosphorylation state of recombiant proteins, comparing them to the sites from meiotic cultures, and initiated work on phosphosite mutants. This study not only deepens our understanding of fundamental biology but also suggests mechanisms behind misexpression of meiotic proteins in cancers.

C 02
Proteomic subtypes of intrahepatic cholangiocarcinoma are linked to patient’s time-to-recurrence

Presenting author:

Freiburg University Hospital, Institute for Surgical Pathology, Breisacher Straße 115a, 79106 Freiburg [DE], tilman.werner@uniklinik-freiburg.de

Author(s):
Tilman Werner, Klara-Luisa Budau, Miguel Cosenza Contreras, Hause Frank, Kurowski Konrad, Pinter Niko, Schüler Julia, Martin Werner, Sigel Carlie, Laura Tang, Peter Bronsert, Oliver Schilling

Intrahepatic cholangiocarcinoma (ICC) is a rare and insufficiently described cancer whose pathological classification remains challenging. Recurrences are frequent, but occur in patient-individual and unpredictable timeframes. In this study, we characterized proteomic profiles of tumors and adjacent tissue from 80 ICC patients via liquid-chromatography mass-spectrometry (LC-MS/MS) in data independent acquisition (DIA) mode to identify predictive markers for the time-to-recurrence (TTR). We found two tumor subgroups: cluster 1 was enriched with extracellular matrix (ECM) components, and cluster 2 showed increased expression of RNA- and protein turnover machinery components. Patients from cluster 1, which also showed increased proteolytic activity in a semi-tryptic analysis, had significantly longer TTRs. An independent survival-statistics model then extracted proteins whose expression correlates with TTR distribution and uncovered similar biological motifs as in the clustering approach as determinants for the TTR. 9 patient-derived ICC xenografts highlighted the role of tumor-stroma interactions. In a principal component analysis based on this multi-species proteomic approach, we observed ECM proteins in association with infiltrating stroma, while tumor proteins were enriched for splicing, translation, and metabolization of RNA. Overall, ICC recurrence appears to shaped by differing protein expression profiles, likely as a result of varying tumor-stroma interactions.

S 02
Maintenance on mitochondrial complexes ensures bioenergetic function in differentiated cells

Presenting author:

Goethe University, Functional Proteomics, Institute for Cardiovascular Physiology, Theodor-Stern-Kai 7, 60590 Frankfurt [DE], wittig@med.uni-frankfurt.de

Author(s):
Ilka Wittig, Juliana Heidler, Heiko Giese, Ralf Brandes

The assembly sequence of mitochondrial complexes has been extensively studied in proliferating cells. These studies mostly reflect de-novo assembly and provide limited information on the dynamics of protein complexes in differentiated cells and tissues. The state of protein complexes in post-mitotic tissues may rather be a balance between biosynthesis and degradation. An important question is whether protein complexes are always assembled de novo or whether remodelling and repair mechanisms maintain mitochondrial function. Complexome profiling combines blue native electrophoresis with quantitative mass spectrometry to identify rare sub-complexes, assembly intermediates and complex remodelling. In this study, we combined complexome profiling and pulse stable isotope labelling of amino acids in cell culture (Pulsed-SILAC) to investigate the turnover and half-life of individual proteins within protein complexes in differentiated post-mitotic C2C12 myotubes. The results represent a comprehensive collection of data on the dynamics of all stable mitochondrial protein complexes. The complete turnover of all complexes of the oxidative phosphorylation system (OXPHOS) takes about one month. We identified subunits of complex I with higher turnover rates in parts of the electron transport modules and service factors involved in these quality control mechanisms to ensure full bioenergetic function in post-mitotic tissues.

G 27
Learning from errors: Deducing the action of aminoglycoside antibiotics from error landscapes

Presenting author:

Max-Planck Institut für Multidisziplinäre Naturwissenschaften, Department for Physical Biochemistry, Am Fassberg 11, 37077 Göttingen [DE], Ingo.Wohlgemuth@mpinat.mpg.de

Author(s):
Ingo Wohlgemuth, Nilanjan Ghosh Dastidar, Nicola Freyer, Christof Lenz, Henning Urlaub, Marina V Rodnina

The accuracy of protein synthesis determines the quality of the proteome and the fitness of the cell. Errors in translation have been associated with aging, cancer and neurological diseases. On the other hand, many antibiotics compromise the fidelity of translation and kill pathogens by disturbing their proteostasis. We use different mass spectrometric workflows to quantify missense errors in cellular proteins and study their impact on protein stability and the fitness of the cell. Recently, our analysis helped to understand the mechanism and exceptional proteotoxicity of aminoglycoside antibiotics (AGAs). AGAs target the bacterial ribosome and induce mistranslation, yet which translation errors induce bacterial cell death was unclear. We found that AGAs stay bound to the translating ribosome and thereby induce strings of consecutive errors, with up to four incorrect amino acids incorporated along a stretch of seven amino acids in a protein. Proteins with such error clusters are enriched in aggregates, indicating stronger protein misfolding. Consistent with the notion that error clusters drive the bactericidal effect of AGA we show that resistance mechanisms towards aminoglycosides can be associated with a dramatic reduction of error cluster formation. Overall, our work shows how the analysis of the microheterogenity of the proteome can help to deduce the cellular action of drugs and to probe the fitness of the cell.

G 07
N-terminomics identifies substrates of the secreted Staphylococcus aureus protease Jep previously missed by classical label-free proteomics

Presenting author:

University Medicine Greifswald, Department of Functional Genomics, Felix-Hausdorff-Straße 8, 17475 Greifswald [DE], hannes.wolfgramm@uni-greifswald.de

Author(s):
Hannes Wolfgramm, Christopher Saade, Leif Steil, Alexander Reder, Stephan Michalik, Christian Hentschker, Manuela Gesell Salazar, Liliane M. Fernandes Hartzig, Patricia Trübe, Barbara M. Bröker, Keenan Lacey, Victor J. Torres, Kristin Surmann, Silva Holtfreter, Uwe Völker

Virulence of Staphylococcus aureus is shaped by a wide range of tightly regulated virulence factors, including several proteases. These proteases act on host factors, contributing to immune evasion and spreading. In addition, there is evidence that secreted S. aureus proteases regulate virulence by processing the pathogen's own virulence factors extracellularly. Protease deletion mutants show altered levels of secreted virulence factors and exhibit hypervirulence in many cases (e.g., Gimza et al., 2021). In our study, we focused on the novel serine protease Jep, which is found almost exclusively in mouse-associated S. aureus strains. We have shown that the deletion of jep in the mouse-associated S. aureus strain JSNZ led to hypervirulence in a murine bacteraemia model. However, using a classical label-free proteomic approach, no differences were found in the secretome pattern of the mutant strain compared to the wild-type strain. This unexpected contradiction was resolved by using N-terminomics, which revealed alterations in the N-termini of a number of secreted proteins, including known virulence factors such as the subunits of LukAB. Our results suggest that the protease Jep influences virulence rather by targeted proteolytic processing of secreted virulence factors than by protein degradation. This example illustrates the power of N-terminomics in the investigation of proteases, to reveal effects that cannot be covered by classical label-free proteomic approaches.

O 05
Analysis of 3CL Protease inhibitors: an automated assay for rapid screening of compounds

Presenting author:

Max von Laue Straße 3, 60438 Frankfurt am Main [DE], jonathan.zoeller@biophys.mpg.de

Author(s):
Jonathan Zöller, Frederic Farges, Barbara Rathmann, Kristina Desch, Joshua Vollrath, Nadide Altincekic, Harald Schwalbe, Julian Langer

This work outlines an innovative approach to investigate potential drugs for the treatment of SARS-CoV-2, the virus responsible for the 2019 novel virus pandemic. Specifically, we developed a MALDI-MS based activity assay, which can be used to rapidly screen for potential inhibitors of the 3CL protease, a key enzyme in the replication of the virus. Our data show that compounds such as a newly identified potential drug named Tamol and Nirmatrelvir, a known inhibitor, strongly inhibit 3CL protease activity. To further investigate the effects of these inhibitors, we utilized HDX-MS and showed that Tamol is likely to cover the surface of domain I of the 3CL protease and that Nirmatrelvir binds strongly to its active site. We also investigated binding of Tamol to the Coronavirus receptor binding domain, and observed only weak interactions. We further acquired preliminary NMR data on the 3CL protease bound to Tamol and observed similar unfolding effects. With effective inhibitors in high demand, further investigation into potential compounds is essential. The developed assays have the potential to significantly expedite the process of finding new compounds that can be used to treat SARS-CoV-2.

Abstract not submitted yet