3rd International Top-Down Proteomics Symposium

Aug 25, 2025, 9:00 AM → Aug 29, 2025, 4:45 PM Europe/Berlin

VMP 6 / Philturm (Lecture Hall D)

Bente Siebels (Universität Hamburg), Hartmut Schlüter (Institute of Biochemistry), Maria Riedner (Universität Hamburg)

Mon, August 25

Short Course 1: Top-Down Proteomics

9:00 AM Proteoforms – Introduction (Hartmut Schlüter)

9:30 AM Analysis of proteoforms in tissues - Sampling and homogenization (Hartmut Schlüter)

10:00 AM Sample preparation for top-down proteomics (Andreas Tholey)

10:30 AM Coffee Break

11:00 AM Liquid chromatography for proteoform fractionation (Hartmut Schlüter)

11:30 AM Concepts and implications for TD/MD MS (Yuri Tsybin)

12:00 PM Lunch

1:30 PM Data processing - concepts and implications for TD/MD MS (Yuri Tsybin)

2:15 PM Mass spectrometry of intact proteins & Fragmentation of proteoforms (Bente Siebels & Maria Riedner)

3:00 PM Coffee Break

3:30 PM Charge Detection for Deep Proteoform Characterization (Neil Kelleher)

Tue, August 26

Short Course 1: Top-Down Proteomics

8:30 AM Biotherapeutics - Analysis of therapeutic proteins (Yuri Tsybin)

9:15 AM Bioinformatics – spectral deconvolution (Kyowon Jeong)

10:00 AM Coffee Break

10:30 AM Bioinformatics of TD/MD MS: use of FLASHApp (Kyowon Jeong, Tom Müller)

11:15 AM Adapting de novo sequence inference to top-down proteomics (David Tabb)

12:00 PM Lunch break

Registration opens

ECR Meeting (Hybrid)

On-site moderators: Mowei Zhou, Kyowon Jeong, Philipp Kaulich, Boris Krichl

Program:
1. Trivia games
2. Introduction to the ECR committee
3. Interlaboratory initiatives of the current ECR (Quantitative, high-throughput TDP: Kellye, Data analysis for Native TDP: Corrine, Ion Mobility TDP: Fabio)
4. Online resource for TDP TopDownVerse https://topdownverse.netlify.app/ : Bryon
5. Online presentation series introducing upper-level graduate students: Kellye

2:00 PM Welcome Coffee

Welcome Session

New Frontiers in Life Sciences - Entering the Proteoform Era

1 New Frontiers in Proteomics - Proteoforms, Proteoform Families, and the Human Proteoform Project

Proteins are the primary effectors of function in biology, and thus complete knowledge of their structure and behavior is needed to decipher function. However the richness of protein structure and function goes far beyond the linear amino acid sequence dictated by the genetic code. Multigene families, alternative splicing, coding polymorphisms, and post-translational modifications, work together to create a rich variety of proteoforms, whose chemical diversity is the foundation of the biological complexes and networks that control biology. "Proteoforms" are the specific molecular forms in which proteins are present in biological systems; only direct analysis of the proteoforms themselves can reveal their structures, dynamics, and localizations in biological systems.

Remarkably, the dominant paradigm of proteomics research, “bottom-up” proteomics, does not identify proteoforms – rather, proteins are enzymatically digested into peptides, whose identification then indicates the likely presence of their parent proteins in the sample. This strategy destroys the information as to what form of the protein the peptide represents, and thus the critical information needed to identify proteoforms is lost. The entire field of Biology is thus attempting to understand life in the absence of the ability to understand the molecules that define life. This limitation of todays technology provides a “grand challenge” to the scientific community, to devise new strategies and approaches that are able to comprehensively and quantitatively reveal the full breadth of the proteome at the proteoform level.

In this presentation I will provide an overview of this interesting problem, along with a variety of new tools and approaches that we and others are developing to address it. Developing the technology to decipher proteoforms, building a comprehensive atlas of proteoforms present in human systems, and eventually deciphering the functional roles they play in normal and disease biology, comprise central elements in the quest to understand human biology.

Speaker: Lloyd Smith (Department of Chemistry, University of Wisconsin - Madison)

2 Digitizing Proteoform Biology with Single Molecule & Single Cell Mass Spectrometry

Since the completion of the Human Genome Project, much has been made of the need to bridge the gap from genes and traits. As a key nexus for the many interacting ‘-omes’ (genome, transcriptome, proteome, metabolome, etc.), the proteome should offer a tight link between genotype and phenotype. Proteoforms, or all of the precise molecular forms of a protein, capture all sources of variability in protein composition (i.e., SNPs, isoforms, post-translational modifications), and thus provide crucial insights into regulation and function. Now, “single ion” mass spectrometry is poised to convert genes to proteoform signatures at a far faster rate. Recently we developed proteoform imaging mass spectrometry (PiMS), with individual ion mass spectrometry. This platform has been extended now to single-cell Proteoform imaging Mass Spectrometry (scPiMS), boosting cell processing rates by >20-fold in the field while detecting proteoforms from single cells.

Speaker: Dr Neil Kelleher

3 Revealing Functional Proteoforms by Native Top-Down Proteomics

Native mass spectrometry (nMS) measures proteins and complexes that are functionally relevant to biology. Top-down proteomics (TDP) reveals identification, sequence, and proteoform information. The combination of these platforms, native top-down proteomics (nTDP), could be ideal for understanding how proteins interact with other proteins (and ligands and cofactors), identifying unknown proteoforms, and gain information on their function at near physiological conditions. We are developing a nTDP workflow based on data independent acquisition (DIA) without on-line chromatographic separation to address two microbial-based projects. Understanding the host-pathogen interface is key to combating antimicrobial resistance. For unfractionated secretomes of model Gram+ pathogenic bacteria, Corynebacterium diphtheriae, a single direct infusion revealed more than 370 unique masses. We identify more than 70 proteoforms, including novel virulence factors and complexoforms reaching 300 kDa. A functional proteomics platform based on slow mixing mode (SLOMO) and DIA-proton charge reduction (PTCR)/higher-energy collisional dissociation (HCD) was developed to determine how they acquire iron during infection. For the second project, we apply nTDP to elucidate the structure and composition of the cellulosome, a massive (0.5-2 MDa), self-assembling, multi-enzyme complex with potential implications to the carbon cycle and sustainable biofuel production. Its function is to break down lignocellulose and other biopolymers. To date, little is known about the specific structure and composition of intact cellulosomes. Preliminary nMS of putative Clostridium thermocellum cellulosomes reveals highly complex spectra. By applying electron-capture charge reduction (ECCR), masses in the 100-300 kDa range were deconvolved. For a 184 kDa complex detected, HCD-based nTDP identified it as a homohexamer enoyl hydratase (29 kDa monomer) complex.

Speaker: Joseph Loo (UCLA)

4 Touching upon the millions of hidden treasures in the plasma proteome

I will describe how innovative techniques in mass spectrometry provide unique novel insights into our humoral immune response. In our body we produce every day huge amounts of antibodies, of which many end up in circulation. Humans can make about trillions of distinct antibody clones, all exhibiting a different sequence, recognizing distinct antigens. We recently developed new LC-MS based antibody repertoire profiling methods for studying immunoglobulins in a quantitative manner. By now, we analysed a variety of samples (sera, milk and saliva) from both healthy as well as diseased donors, allowing us to make some paradigm-shifting observations of which several I will highlight in this talk. Moreover, I will describe how both peptide- and protein-centric approaches on new mass analyser facilitate de novo sequencing, a prerequisite for proper identification of circulating antibodies. Making use of such methods we are now able to identify patient specific antibody responses against diseases specific antigens, which may be considered leads for further therapeutic development. And yes, there are millions of different proteins in our blood.

Speaker: Albert Heck (Utrecht University)

Round Table Discussion

Why do we need a ‘Mars-shot project’ for proteoform research?

Moderators: Guinevere Lageveen-Kammeijer, Manfred Wuhrer

Participants:
Rohan Thakur – Bruker
Jake Melby – AstraZeneca
Max Kraner – NovoNordisk
Alexander Makarov – Thermo
Andreas Huhmer – Nautilus
Michal Sharon (Weizmann Institute of Science)
Neil Kelleher (Northwestern University)

Welcome Reception

6:30 PM Nautilus: Flash Talk

Wed, August 27

New Tools for Proteoform Analysis

5 Advances in hardware design and function of the new timsOmni MS platform

Innovations in mass spectrometry (MS) instrumentation continue to emerge, driven primarily by the need to identify and characterize proteins with greater confidence. Simultaneously, fragmentation schemes are an essential performance component of any MS platform, delivering detailed structural and sequence data necessary for precise identifications. Here we report on the latest advances realized on the new timsOmni platform where novel offline as well as online data dependent acquisition (DDA) experimental workflows are applied for the analysis of different classes of analytes. The diverse operating modes described in this study highlight the exceptional versatility and broad applicability of this novel instrument configuration.

A series of hardware developments are reported including (a) a linear stacked-ring RF ion guide providing efficient desolvation, (b) an ion mobility gate for selecting ions separated in the tims device, (c) a new design of segment Q5 in the Omnitrap accommodating higher electron currents without comprising robustness and (d) an improved AC-ejection method to transfer a wide range of m/z ratios from the collision cell to the TOF analyzer. The range of m/z ratios recorded in single TOF spectra extends from <300 Th to >10,000 Th. An offline ESI source is developed and applied for analysis of protein complexes. Deep sequencing of IgG1 monoclonal antibodies fragmented by various MSn modes is reported. Gas phase reduction of intrachain disulfide bonds is demonstrated for intact mAbs and industrial enzymes using electron-based fragmentation while post translational modifications on histones are identified in MSn experiments. Collision activation followed by mobility separation and electron capture dissociation is performed to map the unfolding process of different protein systems. DDA experiments are performed on a protein standard and digested antibody mixtures. Dynamic control of the accumulation period for quad-selected charge states is shown to improve sequence coverage for all the protein systems and subunits examined.

Speaker: Dr Dimitris Papanastasiou (Fasmatech Science & Technology)

6 Top-down sequencing of intact, modified proteins by timsTOF technology with new multi-modal fragmentation capabilities

We demonstrate top-down sequencing and detailed PTM characterization of intact histone proteoforms using a novel timsOMNI mass spectrometry platform. We prepared well defined acetylated and methylated proteoforms of histones H3 and H4 that were analyzed and sequenced by nanoESI interfaced to a modified timsTOF Ultra mass spectrometer equipped with the omnitrap technology (FasmaTech, Bruker Daltonics). Intact protein sequencing by MS/MS was performed by CID, ECD and combinations thereof. Data analysis was expedited by the Omniscape software (Bruker Daltonics). The timsOMNI platform generated highly informative tandem mass spectra of intact proteins enabling comprehensive sequence analysis, localization of multiple PTM sites and assessment of proteoform heterogeneity.

Speaker: Ole Nørregaard Jensen (SDU Department of Biochemistry and Molecular Biology)

7 Sensitive Top-down Analysis using Spray-capillary-based CE-MS approaches

Quantitative analysis of intact proteoforms in mass-limited, complex samples remains challenging due to the low ion intensity in MS detection and peak overlap caused by insufficient separation. While targeted top-down proteomics methods such as parallel reaction monitoring (PRM) have been developed for LC-MS, they typically require microgram-level sample input, limiting their utility for scarce samples. To overcome this, we recently developed an ultrasensitive spray-capillary-based method that enables ultralow-volume sampling and online CE-MS quantitation of intact proteoforms from picogram-level complex samples such as single cell analysis. To further enhance throughput, we recently introduced a multisegmented injection strategy using this spray-capillary platform. By integrating multisegmented spray-capillary CE-MS with PRM, we achieved highly sensitive and targeted quantitation of intact proteoforms at the attomole level with high throughput (e.g., analyzing 7+ samples in less than one hour). This platform demonstrated high selectivity and specificity, enabling high-throughput characterization and quantification.

Speaker: Si Wu

8 Combining advanced fragmentation techniques and spectral simplification for deep proteoform interrogation

The “proteoform hypothesis” postulates that the proteome-to-phenotype connection is better explained through the characterization of the actual molecules present in a cell or tissue, or proteoforms, than by cataloguing “protein groups” that represent undistinguished molecule ensembles. The top-down (TD) approach to proteomics (i.e., the direct analysis of proteoforms) can theoretically ensure the access to the proteoform landscape of cells and tissues. However, TD is particularly challenging to properly implement, as both intact and fragmentation mass spectra of proteoforms suffer from problems such as signal dilution and ion signal overlap. Additionally, to remain true to its declared mission of molecularly defining proteoforms, top-down mass spectrometry should in principle analyze also typically neglected post-translational modifications such as Cys-linked ones, which remain present on proteins only if disulfide bond reduction is avoided.
We find that the use of ion-ion and photon-ion reactions in the gas-phase leads to extensive sequence coverage of a variety of proteoforms, natural or artificial. Case studies include 66 kDa human serum albumin, which comprises 13 disulfide bonds, and chemically modified proteoforms of antibody-drug conjugates.
We demonstrate that the use of advanced fragmentation methods such as activated ion electron transfer dissociation (AI-ETD), where low-energy IR photons are used to denature protein cations while ETD takes place, are beneficial for both increasing the number of identified backbone cleavages and sequencing disulfide-protected regions that remain otherwise uncharacterized. We also show how incorporating collisional activation of product ions post AI-ETD (referred to as AI-EThcD) further increases proteoform sequence coverage.
Finally, we show that reducing signal overlap of product ions via ion-ion reactions, specifically proton transfer charge reduction (PTCR), does not only give access to additional sequence information generated by these fragmentation methods, but it also dramatically facilitates the interpretation of complex mass spectra, substantially reducing the number of false positive matches.

Speaker: Luca Fornelli (University of Oklahoma)

9 Mass-Invariant Log-Transformed Mass Spectra Enable De Novo Sequencing and Internal Calibration of Intact Proteins

Most top-down proteomics workflows rely on deconvolution of intact and fragment ion m/z values using modeled isotope distributions, typically via an “averagine” approximation. This step often limits accuracy: poor fits to distorted isotope patterns can lead to incorrect monoisotopic mass assignment, widened mass tolerances, and inflated false discovery rates. To address these limitations, we have developed a framework for de novo sequencing and internal calibration that operates entirely in natural log-transformed m/z space—eliminating the need for monoisotopic mass determination.

By transforming spectra to ln(m/z − q), where q is the charge carrier mass, peaks arising from the same analyte mass align along a predictable pattern defined solely by charge state—a principle formalized by Jeong et al. in the FLASHDeconv algorithm (2020). This mass-invariant spacing can be used to assign charge states, pair isotopologues, and perform internal calibration without averagine-based fitting. Calibration is achieved by optimizing the B coefficient in the Ledford equation until observed peaks align with the expected −ln(c) spacing. Sequence tag inference is performed by comparing log-transformed peak positions from consecutive fragment ions to expected values based on known residue mass differences. When observed ln(m/z − q) values match those predicted for a given residue across multiple isotopologues and charge states, the corresponding mass difference can be confidently assigned—even from a single scan.

This method was applied to 21 T FT-ICR MS/MS spectra of intact proteins, achieving sub-ppm agreement between predicted and observed values without spectral averaging. Internal calibration improved mass accuracy of myoglobin from 6.9 ppm RMSE to 0.8 ppm. Notably, near-isobaric residues such as lysine and glutamine were resolved at high charge state, and proteoform families were identified from MS¹ data using log-space mass differences alone. This database-independent, calibrant-free framework enables high-accuracy proteoform analysis and significantly improves the robustness and resolution of top-down de novo sequencing.

Speaker: Lissa C. Anderson (National High Magnetic Field Laboratory, Florida State University)

10:00 AM Coffee Break

Lightning Talks

10 Properties, Origin, and Reproducibility of Truncated Proteoforms Across Top-Down Proteomic Studies

Top-down proteomics (TDP) offers a powerful approach to identifying intact proteoforms, inherently providing information about protein modifications. Truncated proteoforms are among the most frequently observed modifications in TDP studies. Here, we selected fifty datasets, including over 140,000 proteoforms, published over the past decade, spanning various organisms, sample preparation approaches, data acquisitions, and data analysis pipelines, and investigated the reported proteoforms. On average, across all studies, 70% of the proteoforms were truncated, and only 30% were identified as full-length proteoforms (including those with only N-terminal methionine excision). Only 16% of the exclusively N-terminally, 5% of the C-terminally, and 1% of the N- and C-terminally truncated proteoforms have been described in the UniProt database, highlighting that the biological function of most truncations is unknown. To understand cleavage patterns more clearly, we determined the amino acids N- and C-terminally located from the truncation sites. We found truncation sites to be very diverse, with specific datasets showing unique patterns. Several truncation sites indicated artifacts introduced during sample preparation (e.g., between the aspartate-proline bond due to sample heating), mass spectrometric analysis (e.g., N-terminal to proline residues due to in-source fragmentation), or data analysis (e.g., due to database and precursor tolerance settings). Moreover, substantial differences between samples of the same organism but different tissues or growth conditions were also observed. Importantly, our analysis revealed specific protein termini not linked to artifactual cleavages that were consistently reported across numerous studies, implicating these proteoforms to have biological significance. This includes previously unannotated mitochondrial signal peptide sites and cleavages with protein domain or structural specificity. In conclusion, this study provides a comprehensive overview of truncated proteoforms identified in recent TDP studies, highlighting both methodological and biological influences. We believe these results can also serve as a resource for other scientists to investigate non-canonical termini from proteins of interest.

Speaker: Dr Philipp T. Kaulich (Systematic Proteome Research & Bioanalytics, Institute for Experimental Medicine, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany)

11 Novel bridged hybrid monolithic columns combined with mass spectrometry for top-down proteomic analysis

Top-down proteomics could offer comprehensive investigations of proteoforms. However, it is greatly challenged by the co-elution of intact proteins which results in overlapped mass spectra. Hence, highly effective protein separation to reduce the co-elution of intact proteins from complex samples is of vital importance.
The ethane-bridged hybrid monolithic column with homogeneous macropores of 1.1 μm and large mesopores of 24 nm was prepared for protein separation with high peak capacity of 646 within 240-min gradient. Based on MS/MS analysis, 959 proteoforms corresponding to 263 proteins could be unambiguously identified from E. coli lysates in a single 240-min run. Furthermore, 347 large proteoforms with Mw higher than 30 kDa were detected in the single 75-min run. Besides, 6264 proteoforms corresponding to 885 proteins were identified from THP-1 cells induced by LPS.
The amine-bridged hybrid monolithic column with unique macropores was prepared and coupled to MS for analysis of membrane proteoforms. Due to its unique macroporous structure and secondary amino groups in the framework, the column possessed fast mass transfer, low non-specific adsorption, and electrostatic repulsion to membrane proteins, thus greatly reducing peak broadening and outperforming traditional reversed-phase columns in top-down characterization of membrane proteoforms. With this column, a total of 3100 membrane proteoforms were identified in the mouse hippocampus. The proteoform information was integrated into the interaction network of membrane protein complexes involved in oxidative phosphorylation processing, uncovering more detailed molecular basis and interaction in the biological processes.
Furthermore, highly sensitive top-down proteomic analysis of LCM slices was developed based on the narrow-bore amine-bridged hybrid monolithic column with low non-specific adsorption. Integrated with MALDI MSI, high-throughput spatially resolved proteoform analysis were achieved, yielding 366 annotated proteoform images from the mouse brain and revealing 14 differential proteoforms in the subiculum region associations with amyloid-β pathology in AD.

Speaker: Dr Yu Liang (Dalian Institute of Chemical Physics, CAS, China)

12 Spatial Phosphoproteomic Profiling of Murine Heart Reveals Region-Specific Functions via TiO₂ Enrichment Optimized for Laser-Capture Microdissected Samples

Phosphorylation-mediated signaling dynamics across spatially distinct cardiac regions remain poorly understood due to limited technical capacity for deep and sensitive analysis of minute samples. Here, we present an optimized TiO₂-based micropipette tip method for deep phosphoproteomics, achieving high sensitivity (12,117 class I phosphosites from only 10 µg HeLa peptides) and reproducibility. Applying this to laser-capture microdissected mice myocardial regions, i.e. left/right atria (LA, RA), left/right ventricles (LV, RV), interventricular septum (IVS), apex (APEX), and aortic valve (AV), we quantified 1,000–2,000 class I phosphosites per region (e.g., 1,050 in AV, which has an area of only 0.2 mm²). Principal component analysis revealed distinct phosphoproteomic clustering aligned with anatomical positions, surpassing proteomic resolution. Functional enrichment uncovered region-specific functions: APEX and ventricles exhibited phosphorylation signatures linked to muscle contraction, while AV was enriched in cell junction and polarity. Metabolically, the LV demonstrated phos-phorylation patterns linked to energy metabolism, whereas LA showed enrichment in RNA processing. RA was pertinent to cellular component biogenesis and chromatin organization. This spatially resolved phosphoproteomic atlas elucidates func-tional specialization across cardiac subregions, establishing a molecular foundation for investing region-specific cardiac pa-thologies. Our approach addresses critical technical limitations in low-input phosphoproteomics while advancing under-standing of cardiac spatial heterogeneity at the post-translational level.

Speaker: Dan Zhao

13 FLASHApp: Interactive Data Analysis and Visualization for Top-Down Proteomics

Introduction
Top-down proteomics (TDP) is increasingly being applied in proteoform-resolved biomedical and clinical research. The complexity of TDP data demands flexible visualization tools integrated with analysis workflows to streamline interpretation and validation. Existing tools often lack adaptability and interactivity, requiring complimentary manual analysis to generate publication-ready results and figures. This added layer of manual intervention impacts reproducibility, posing a significant challenge to consistent scientific outcomes.
FLASHApp addresses these challenges by providing an interactive solution for TDP data analysis and visualization. As a free, open-source, web-based application it is accessible on any modern computer at https://www.openms.org/FLASHApp/.
Methods
FLASHApp is based on the OpenMS Streamlit template. It supports methods involving TDP tools for spectral deconvolution, quantification, and characterization of proteoforms. In addition to being publicly accessible via the OpenMS website, FLASHApp can easily be hosted in-house (e.g. by core facilities) using a containerized image. A Windows installer eases offline execution for non-bioinformaticians.
Results
Upon entering FLASHApp, a new workspace is automatically created and embedded within the website URL, enabling users to bookmark their session, revisit analyses at a later time, and share results with collaborators.
The app's sidebar organizes common TDP tasks into dedicated sections, each guiding users through uploading input files, configuring parameters, executing tools, visualizing outputs, and downloading results. Tailored, publication-ready visualizations presented in a user-configurable layout address the specific requirements of each task, enhancing both interpretability and efficiency of data analysis. The interactive nature of these visualizations allows dynamic adjustments, such as modifying fragment ion matching tolerances.
Conclusion
FLASHApp provides modular, interactive visualizations specifically designed for TDP data analysis, improving usability, reproducibility, and accessibility through both web-based and local setup. These features position FLASHApp as a flexible, user-friendly platform that streamlines TDP workflows, facilitates collaboration through shareable URLs, and aids reproducible scientific discovery.

Speaker: Tom David Müller (University of Tübingen)

14 Metabolomics and proteomics reveal the inhibitory effect of Lactobacillus crispatus on cervical cancer

Cervical cancer remains a significant global health issue due to its high morbidity and mortality rates. Recently, Lactobacillus crispatus has been recognized for its crucial role in maintaining cervical health. While some studies have explored the use of L. crispatus to mitigate cervical cancer, the underlying mechanisms remain largely unknown. In this study, we employed non-targeted proteomics and metabolomics to investigate how L. crispatus affects the growth of cervical cancer cells (SiHa) and normal cervical cells (Ect1/E6E7). Our findings indicated that the inhibitory effect of L. crispatus on SiHa cells was associated with various biological processes, notably the ferroptosis pathway. Specifically, L. crispatus was found to regulate the expression of proteins such as HMOX1, SLC39A14, VDAC2, ACSL4, and LPCAT3 by SiHa cells, which are closely related to ferroptosis. Additionally, it activated the tricarboxylic acid (TCA) cycle in SiHa cells, leading to increased levels of reactive oxygen species (ROS) and lipid peroxides (LPO). These results revealed the therapeutic potential of L. crispatus in targeting the ferroptosis pathway for cervical cancer treatment, opening new avenues for research and therapy in cervical cancer.

Speaker: Lingyan Zhong

15 SEC-complex-down approaches with functional O2-affinity assay: Correlation between the higher order structure of bird hemoglobin homologues and their function.

Hemoglobin is a tetrameric protein responsible for the blood oxygen transport. Any abnormalities in hemoglobin structure can lead to serious health outcomes. Although very well characterized in humans, it has been scarcely studied in the case of birds. Some studies have been carried out at the globin level without providing further evidences about the native structure of the complex. In this context, we propose for the first time the combination of size-exclusion chromatography with complex-down mass spectrometry for the straight correlation of binding stoichiometry of subunits, their primary structure, and the identity of different cofactors. Complex-up analysis were carried out to decipher the identity of the individual constituents of each tetramer population by providing increased energy in the mass spectrometer ion source. Thus, it was concluded that mass differences between the three tetramers were due to the substitution of alpha subunits. In addition, one cofactor was also identified for the first time linked to the tetramers.
Information about subunit sequence characterization and cofactor identification were afforded through SEC-complex-down approach (pMS3) to isolate and fragment the different constituents. Fragmentation of subunits was achieved by combining various fragmentation methods leading to an overall sequence coverage of 98, 94, and 96% for aA, aD, and b subunits, respectively. The pMS3 approach allowed to determine the presence of IP5 cofactor, which is known to regulate 02-hemoglobin affinity in birds.
Finally, the three tetramers were collected separately upon SEC separation to record 02-affinity data for each tetramer. The functional data clearly showed affinity differences between the three populations, clearly pinpointing for the first time that bird hemoglobin affinity correlated with the number of aD subunits in the tetrameric structure. Altogether, the results from the SEC complex-down analysis in combination with O2-affinity data could find application in evolution analysis, environmental adaptation, or different clinical contexts.

Speaker: Léa Letissier (LSMBO - CNRS (Université de Strasbourg))

16 Multi-dimensional High-Throughput Molecular Glue Screening via Gas Phase Affinity Selection Native Mass Spectrometry and Cryo-EM Analysis

Targeted protein degradation of undruggable proteins is transformative in drug discovery. Molecular glues (MGs) enhance weak interactions between targets and E3 ligase. Native mass spectrometry (nMS) identifies E3-MG-target complexes directly, but manual sample preparation limits throughput.

This study demonstrates high-throughput MG screening using nMS for WEE1 binding to CRBN-DDB1, enabling multiplex screening of over 2,500 compounds per day. Gas-phase ligand release and fragmentation help identify unknown binders, and cryo-electron microscopy (EM) analysis characterizes ligand-bound complexes.

We compressed 96 compounds into 24 mixtures and used a SEC column for rapid online buffer exchange. LC-MS screening of all 96 compounds took under an hour, achieving throughput of over 2,500 compounds per day. Strong ternary complex formation between CRBN-DDB1 and WEE1 was observed in 4 of 24 mixtures, with 2 additional samples showing moderate binding. Identifying individual binders within mixtures was challenged by compound multiplexing, native adduct interference, and non-specific interactions.

To resolve this, ternary complexes were isolated in the quadrupole and subjected to collision-induced dissociation. Released binders, potentially uncharged in the gas phase, were detected via polarity switching. MS2 analysis of low m/z ions enabled accurate mass determination and comparison with the compound library. Ligands with unmatched masses were classified as "Unknown" and further analyzed by MS3 fragmentation for structural elucidation.

This workflow uses MS1 to detect intact complexes, MS2 to identify bound ligands, and MS3 to characterize unknowns, increasing throughput and specificity in MG screening. This approach confirmed 16 of 96 compounds as potential MGs, with varying binding strengths.

Selected hits were further characterized using cryo-EM, yielding high-resolution structures of WEE1-MG-CRBN-DDB1 ternary complexes. These structures reveal how MGs mediate and stabilize protein-protein interactions, offering critical insights to guide drug design and optimization.

Speaker: Amanda Lee (Thermo Fisher Scientific)

17 Legionella effector AnkX puts the brakes on IMPDH2 filaments

Bacterial pathogens often utilize post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires' disease, secretes the enzyme AnkX, which is known to use cytidine diphosphate-choline to post-translationally modify the human small G-protein Rab1 with a phosphocholine moiety. Later during the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism of Rab1's post-translational modification has been extensively studied by us, we have identified a previously unknown host target protein of AnkX: the metabolic enzyme IMPDH2. IMPDH2 catalyses the conversion of IMP to XMP, which is crucial for guanine nucleotide biosynthesis. Each IMPDH2 monomer features a catalytic domain and a regulatory Bateman domain, which binds ATP and GTP to regulate enzymatic activity and filament formation. IMPDH2 reversibly assembles into filaments in cells, which is thought to provide an additional layer of regulation. Currently we investigate the molecular mechanism and consequences of IMPDH2 phosphocholination by AnkX. Mass spectrometry revealed the modification site within IMPDH2 and demonstrated that, in contrast to Rab1, Lem3 cannot reverse the modification of IMPDH2. While the modification does not alter the catalytic activity of IMPDH2, it disrupts filament formation, thereby impairing a key regulatory mechanism of enzyme function.

Speaker: Marietta Sandkamp-Kaspers (Universität Hamburg)

18 Top-Down Mass Spectrometry of a Clinical Antibody Light Chain Using the Omnitrap-Orbitrap-Booster Platform

The Omnitrap-Orbitrap-Booster (OOB) mass spectrometry (MS) platform with enhanced high-resolution, fragmentation and data acquisition capabilities, was developed to advance top-down (TD) MS analysis of proteins. It integrates a multimodal tandem mass spectrometry (MS/MS) ion trap system (OmnitrapTM) offering a wide range of fragmentation methods, a high-resolution Orbitrap Fourier transform mass spectrometer (FTMS), and a high-performance data acquisition system (FTMS Booster) to improve fragmentation efficiency and spectral quality by increasing the signal-to-noise (S/N) ratio of product ions. In this study, we evaluate the OOB platform for electron capture dissociation (ECD)-based TD MS analysis of a clinical multiple myeloma antibody light chain extracted from the patient’s (P15) urine sample, benchmarking its performance against the “gold-standard” electron transfer dissociation (ETD)-based TD MS on an Orbitrap EclipseTM. These analyses were performed with online coupling to a LC system operating in nanoflow mode. Comparable sequence coverage was obtained for single precursor charge state analysis between ECD-based TD MS on OOB and ETD-based TD MS on EclipseTM (68.2% vs. 74.3% respectively). ECD showed a lower spectral peak density and reduced redundancy of product ions. Moreover, the analysis of multiple precursor charge states (15+ to 19+) sequentially across 5 runs of LC-MS/MS on the OOB platform improves the sequence coverage to 93%, indicating its suitability for comprehensive characterization of protein. Thus, this study uniquely establishes the OOB platform as a highly powerful and efficient system for TD MS of proteins.

Speaker: Marie Yammine (INSTITUT PASTEUR - CNRS)

19 X-ray spectroscopy meets native mass spectrometry: probing gas-phase protein complexes

X-ray activation and dissociation of proteins and their non-covalent assemblies may elucidate structural and functional details complementary to established top-down mass-spectrometry techniques. This is attributed to the rapid, site-specific ionization of atoms within biomolecules. Our research group conducted proof-of-concept experiments exploring X-ray activation of samples with masses ranging from small 17 kDa monomeric proteins to large 800 kDa non-covalent protein complexes at synchrotron (PETRA III) and free-electron laser (FLASH2) facilities. A quadrupole time-of-flight mass spectrometer, adapted for high-mass analysis, was further modified to enable photon-ion interactions. Native proteins and their complexes were introduced into the gas phase via nano-electrospray ionization and exposed to either extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their native folded state or following gas-phase collision-induced activation. The resulting effects—fragmentation, dissociation, or enhanced ionization—varied depending on biomolecule size and activation method. We also explored the integration of ion mobility to enhance structural separation prior to X-ray probing. Within the rapidly evolving domain of X-ray technologies, the activation of large proteins and their complexes via X-rays holds significant potential for advancing top-down analysis and structural biology research.

Speaker: Dr Jocky Chun Kui Kung (Centre for Structural Systems Biology)

20 A prototype TIMS-FT-ICR MS instrument capable of deep characterisation of complex samples and biomolecules

Trapped ion mobility spectrometry (TIMS) spatially separates ions when suspended in a high-pressure region between a retarding electric field gradient and constant gas flow from the atmospheric pressure inlet of the mass spectrometer. TIMS allows high resolution separation of ions/isomers/conformers with varying duty cycles in a relatively small device (<10cm). TIMS is also particularly well suited to slower scan speed instruments such as FT-ICR MS via the use of gated-TIMS. Herein we show initial data from a novel, fully integrated gTIMS-FT-ICR MS instrument, optimisation of this marriage, and its application to key areas such protein conformer-selective ExD MSMS.

The new gTIMS-MRMS system codenamed MATCH was built by combining commercial SolariX MRMS and TIMS-ToF Flex (Bruker Daltonics, Germany) instruments into a prototype. The front of the TIMS Tof Flex , including dual ESI+MALDI source, accumulation during separation dual-TIMS cartridge, mass-resolving quadrupole, and collision cell were combined via a custom transfer region with the back-end elements of the SolariX MRMS system; UHV-isolating beam valve, ~1m transfer hexapole to inject ions through the fringe field of the 7T superconducting maxwell magnet, Paracell detector with electron dissociation (ExD) cathode, and 2-omega detection.

Native MS analysis of proteins was conducted on model isolated proteins to investigate the ability of gTIMS to separate and analyse proteins of interest without significantly affecting structure. Match was developed to use an ultra-low energy transfer and storage energy gradient throughout the TIMS separation, storage, and transfer processes, enabling batch-accumulation of conformer ensembles selected for enhanced dynamic range of downstream MS/MS. Collision-induced unfolding (CIU) was achieved using gradient potentials between the accumulation and analysis portions of the TIMS funnel. Subsequent IMS and quad selections of conformers of interest along the CIU profile followed by ECD MS/MS in the ICR cell revealed changes in fragmentation patterns depending on the conformer selected.

Speaker: Christopher Wootton (Bruker Daltonics)

21 Pin-pointing phosphorylation-dependent Pin1 binding to a cytoskeletal protein altered in Alzheimer's Disease using structural mass spectrometry

Abnormal protein phosphorylation is a fundamental trigger in the pathogenesis of Alzheimer’s Disease, leading to the formation of neurofibrillary tangles. Thus, molecular determination of the critical factors in controlling phosphorylation is in high demand. Pin1, a cis-trans prolyl isomerase has recently been implicated in Alzheimer’s Disease progression. Moreover, Pin1 specifically targets phosphoproteins, regulating their function. Here, we utilise a combination of native MS and top-down MS to reveal a novel interaction between Pin1 and the Collapsin Response Mediator Protein-2 (CRMP2); a protein found hyperphosphorylated alongside tau within neurofibrillary tangles. Using native mass spectrometry, we show that Pin1 binds specifically to the disordered C-terminus of CRMP2 in a phosphorylation-dependent manner. Hydrogen-deuterium exchange mass spectrometry experiments further localised this binding site to the WW-domain of Pin1. Together, the data highlights how mass spectrometry has been utilised to provide novel insight into the regulatory role of Pin1 in a disease-relevant context.

Speaker: Mr Nikolas Brooks (School of Biosciences, University of Birmingham, Birmingham, B15 2TT)

22 Enhancing Drug-Payload Localization in Antibody-Drug Conjugates with a Middle-Down Approach Utilizing Proton Transfer Charge Reduction on an Orbitrap Ascend BioPharma Tribrid Mass Spectrometer

Monoclonal antibodies (mAbs) have revolutionized biotherapeutics, offering effective treatments for various diseases. Antibody-drug conjugates (ADCs) combine mAbs’ specificity with potent cytotoxic drugs linked via Lys- or Cys-conjugation, targeting malignant cells. However, heterogeneity in payload attachment challenges ADCs' safety and efficacy. We employed a middle-down (MD) mass spectrometry (MS) approach to investigate a SiLu ADC mimic with variable drug-to-antibody ratio (DAR) using the Orbitrap Ascend BioPharma Tribrid mass spectrometer. By integrating native MS of 100 kDa F(ab’)2 subunits with disulfide-reduced 25 kDa subunit analysis under denaturing conditions, we achieved thorough ADC characterization and precise localization of payload conjugation sites. Native MS analysis of F(ab’)2 subunits isolated specific DARs with high purity, determining major payload occupancy combinations for DARs 2-8. Our disulfide-reduced subunit analysis aimed to localize payloads on Fd’, which has up to three conjugation sites. While higher-energy collisional dissociation (HCD) and ultraviolet photodissociation (UVPD) provided limited diagnostic ions, Electron Transfer Dissociation (ETD) and ETD followed by supplemental HCD energy (EThcD) localized the conjugation site in the most abundant Fd’ species with a single payload at the MS2 level. Minor single payload species and double payload species required spectral decongestion via proton transfer charge reduction (PTCR) at the MS3 level for higher ion count and sequence coverage. EThcD MS2-PTCR MS3 uniquely localized payloads for all species combinations, achieving ~60% sequence coverage. This approach provided unambiguous localization of drug-payload attachment sites through PTCR following EThcD on the Orbitrap Ascend BioPharma MS.

Speaker: Rachel Grady (Thermo Fisher Scientific)

23 Digital Membrane Chromatography — A New Way to Rapid Antibody Purification for Top-Down MS

Speaker: Kilian Müller (UKE)

12:00 PM Lunch Seminar (Agilent Technologies)

Poster Session 1

Native MS & Protein Complexes

24 Uncovering the unique properties of circulating proteasomes: A mass spectrometry perspective

Proteasomes are well-known mediators of intracellular proteostasis, yet their role in the extracellular space remains largely unexplored. Our recent study investigates the molecular architecture and functional specialization of freely circulating 20S proteasomes (c20S) in the bloodstream. Leveraging a CRISPR-engineered transgenic mouse model, we purified c20S complexes and applied a combination of native and top-down mass spectrometry to dissect their structural and compositional features. Our analyses revealed that serum proteasomes are predominantly uncapped 20S complexes, assembled intracellularly and exported to the blood. Native MS confirmed their intact assembly, while top-down MS identified a suite of post-translational modifications—including cysteinylation, glutathionylation, and truncations—that distinguish c20S from intracellular proteasomes. These extracellular complexes are enriched in immunoproteasome subunits and display enhanced caspase-like activity, indicating specialized roles in the blood environment. Together, these results showcase the power of integrative MS approaches in characterizing proteasome proteoforms and underscore the unique biology of circulating proteasomes, with potential implications for diagnostics and extracellular proteostasis.

Speaker: Michal Sharon (Department of Biomolecular Sciences, Weizmann Institute of Science)

25 Flying viruses – mass spectrometry meets X-rays

Viruses affect basically all organisms on earth. Some are detrimental to human development as we experienced during the COVID-19 pandemic, whereas those targeting pathogenic bacteria or crop pathogens can be beneficial for us. An integral part of icosahedral viruses is the capsid protein shell protecting the genome. Many copies of the capsid protein often self-assemble into shells of defined size. Low binding affinity of individual subunits allows efficient assembly and gives rise to highly stable particles. However, modifications can alter their size. Proteoforms also matter in viral replication, e.g. in polyprotein processing, or antigenicity of the glycoproteins.
Capsids and viral proteoforms can be studied by native mass spectrometry (MS), a single molecule like approach, in terms of stoichiometry, dynamics, assembly pathways and stability revealing coexisting states. However, the structural resolution provided is limited. Therefore, we built a prototype native mass spectrometer in the MS SPIDOC project to deliver select species to X-ray sources for gas phase SAXS and single particle imaging. First experiments reveal good performance of the MS setup.

Speaker: Charlotte Uetrecht (CSSB/DESY/University of Lübeck)

26 Filling the Structural Knowledge Gap in Protein Design via Native Mass Spectrometry

Enzymes are powerful molecules for highly efficient and sustainable chemical synthesis. However, natural enzymes often have limitations and require optimization for large-scale industrial applications. Propelled by artificial intelligence, computational advances such as AlphaFold opened new venues for structure-based enzyme design. However, experimental validation still largely relies on high throughput screening (HTS) for functional and phenotypical data. HTS methods for structural screening only offer limited data and cannot readily resolve multi-component or heterogeneous systems. Only a few successful candidates will have the chance for in-depth structural biology analysis to interpret the mechanism due to the low throughput.
Furthermore, the majority of the “failed” designs can’t be easily formulated into meaningful knowledge to improve the computational models, leading to significant losses of research resources. Native mass spectrometry (native MS) can characterize molecular structures and interactions with fast speed, potentially filling the missing mechanistic knowledge in HTS methods. When combined with top-down MS techniques (native top-down, complex-up, etc.), subunit and residue level information can also be extracted. Recently, native MS have been integrated with computational drug design workflows for finding drug candidates. But more fundamental studies and method developments are still needed to accurately define the correlation of subtle structure features with native MS data, especially for weak interactions.
Herein we selected an artificial triplet photoenzyme RamR with two of its tailored triplet quenchers, and investigated their interactions. The two molecules has different potency in enzyme inhibition, but only differ by the presence/absence of a carboxyl group. Our preliminary data suggested the enzyme-inhibitor interaction was significant but likely non-selective on protein surface. Further experiments are ongoing to examine the enzyme-inhibitor interaction in presence of the native substrate. In summary, the native MS data improve our understanding of the biophysical mechanism of the molecular interactions, and help inform rationale design for enzymes.

Speaker: Mowei Zhou (Zhejiang University)

2:40 PM Coffee Break

Databases & Bioinformatics

27 Replacing “OR“ Logic and Mass Accuracy with “AND/OR“ Logic and Mass Resolving Power, as the Basis for Peak Assignment in Top-Down Mass Spectrometry Data.

This talk concerns how to assign a peak in a fragment ion mass spectrum to a polypeptide sequence. This task is recognized to be problematic for some internal fragment ions, and as we will show, is problematic for all ion types (e.g., terminal fragments). Most experimental mass (peak) to theoretical database entry (molecule) correlations are delivered as a biproduct of automated peptide/proteoform-spectral (PSM) matches. Unfortunately, peak assignments are not what PSMs were designed to do. There are conditions where it is fair to assume—as many do—that PSM peak assignments are correct. We show these conditions involve sparsely populated experimental data generated, for example, with prior MS technology. We propose and demonstrate that assigning fragments ions from rich, e.g., modern, mass spectra, including from TDMS, requires changing the search paradigm from “OR” to “AND/OR” logic for assigning a given peak to related database entries, as well as moving from the use of mass accuracy-related search space to a mass resolution-related search space.

Speaker: Jeffrey Agar (Northeastern University)

28 Advancing Top- and Middle-Down Antibody Analysis Using Simulated FTMS Datasets

The structural complexity and heterogeneity of monoclonal antibodies (mAbs) continue to pose analytical challenges. Over the past three decades, top-down (TD) and middle-down (MD) MS approaches have become powerful tools for characterizing intact antibodies and their subunits [1]. These methods are routinely applied in our CRO operations to complement intact mass and bottom-up proteomics, particularly for resolving complex structural questions in real-world mAb samples. However, despite major advances in instrumentation and data analysis, the structural detail gained from current TD/MD MS workflows remains limited.

A key obstacle to improving TD/MD MS bioinformatics is the lack of standardized, annotated benchmark datasets. To address this, we launched the ProteoGold initiative. It is focused on generating high-quality, in silico FTMS datasets that mimic the complexity of real experimental spectra. Using the proprietary FTMS Simulator (Spectroswiss), we produce isotopically resolved datasets based on user-defined instrument settings and known protein sequences [2]. The simulator is available as a desktop application for full-spectrum simulations and as a web-based platform at www.peakbypeak.com for real-time isotopic modeling, profile-mode spectrum generation, and hybrid server-side processing.

As a proof of concept, we generated a simulated ETD TD MS dataset of carbonic anhydrase II, modeled after data from a 21 T FT-ICR MS at the MagLab [3]. Additional simulations include TD/MD MS datasets of mAbs and subunits acquired on various FTMS platforms, such as Orbitraps. For example, we simulated data from an antibody light chain analyzed on the Omnitrap-Orbitrap-Booster (OOB) platform at the Institute Pasteur, Paris [4].

These datasets form the foundation of the ProteoGold repository, supporting benchmarking, deconvolution evaluation, improved ion assignment, and driving innovation and education in TD/MD MS analysis.

1. Khristenko, et al., Mol. Cell Prot., in press
2. Nagornov, et al., JASMS (2022) 1113-1125
3. Weisbrod et al., JASMS (2017) 1787–1795
4. Garcia et al., submitted

Speaker: Yury Tsybin

29 Computational methods in top-down proteomics to address challenges in proteoform analysis

Top-Down Proteomics (TDP) has emerged as the dominant method for elucidating the intricacies of proteoform diversity, providing insights crucial for understanding biological processes. With development ranging from sample preparation to instrumentation, there has been a notable increase in research endeavors adopting and developing different TDP protocols that suit the objectives of the studies. Moreover, the information density within TDP data sets have grown dramatically, and more TDP data sets are being deposited in the public repository like PRIDE.

Fully realizing the potential of TDP for proteoform resolved analysis requires robust, flexible, and reproducible computational methods capable of handling the complexity of analytes (proteoforms) and data (spectra), while also accommodating the different requirements inherent in each experimental protocol. Due to distinct characteristics (e.g., complexity of ion signals), the computational tools used in the well-established field of bottom-up proteomics (BUP) cannot be readily adopted for TDP; dedicated methods are still demanded for the data analysis, data acquisition, and signal processing.

In this talk, I will introduce our contribution to the field of computational TDP, which include various computational methods such as deconvolution and quantification. I will provide a concise overview of the main concepts and core results of each method, and outline the future directions our group intends to pursue. Finally, the current project for the proteoform identification and characterization method that push the boundaries of the existing search engines will be discussed.

Speaker: Kyowon Jeong (University of Tübingen)

30 TDAuditor assesses deconvolution quality for the Blood Proteoform Atlas

The new TDAuditor software generated quality metrics across the 1551 RAW files of the CTDP Blood Proteoform Atlas (BPA). The algorithm incorporates both spectral clustering and de novo sequence tagging. Multi-threading makes it possible to evaluate 100 mzMLs per minute on a standard desktop PC. The software produces reports in tab-delimited text and mzQC (JSON) formats.

The metrics reported by TDAuditor from the BPA illustrate substantial differences in deconvolution among ProSight PD's "Xtract", TopPIC Suite's "TopFD", and OpenMS' "FLASHDeconv." The precursor charge states and number of masses produced from MS/MS scans have only superficial agreement. A re-identification of all 1551 BPA experiments via TopPIC Suite shows that TDPortal and TopPIC are using very different search spaces, making identifications even more diverse among pipelines than deconvolution differences would suggest.

The advanced signal processing in TDAuditor seeks redundancy among MS/MS scans and attempts to predict identifiability on the basis of deconvolution outputs. Spectral clustering compares deconvolved mass lists for every pair of MS/MS scans in a given mzML file. The resulting graphs of MS/MS relationships illustrate the redundancy of top-down MS/MS measurement and reveal the high-level structure of a top-down experiment. Sequence tagging attempts to infer contiguous amino acid sequences from deconvolved MS/MS scans. The length of the longest tag from an MS/MS scan can predict its identifiability; researchers can use these values to find the "best MS/MS that our search didn't identify."

TDAuditor offers top-down researchers a much more complete appraisal of the LC-MS/MS experiments they generate, and the software is free to use and to modify.

Speaker: Prof. David Tabb (University Medical Center of Groningen)

31 The Implementation of Open Science Practices Can Enable A Faster Development Of Top-Down Proteomics

Open data and science practices, including e.g. the FAIR (Findable, Accessible, Interoperable and Reusable) data principles, are widely implemented in the life sciences. Although this process started years later in proteomics than for other more established omics approaches (e.g. genomics and transcriptomics), their implementation in the field have enabled spectacular advances e.g. in analytical and computational data workflows, including the integration of large amounts of proteomics data in bioinformatics data resources. In the concrete case of top-down proteomics (TDP) and proteoform-centric data, the implementation of open science practices has been more limited due to different reasons, and there is the need for some key new developments. In my view, one of the main priorities of the TDP field should be to fully endorse and implement them to enable new approaches that would help to develop the field faster and a wider dissemination of the outputs of the field. In my talk, I will describe some concrete needs and ideas in this context for TDP and proteoform data

Speaker: Dr Juan Antonio Vizcaino (European Bioinformatics Institute (EMBL-EBI))

4:40 PM Break

Minimum Information Describing A Proteoform (MIDAP) Proteoform Atlas Compendium System (PACS)

7:00 PM Conference Dinner

Meet us at the Rickmer Rickmers!

Thu, August 28

Sample Preparation & Separation Technologies

32 Enabling High-Throughput Proteoform Analysis via Gel-Based Sample Pre-Fractionation with PEPPI-SP3

Achieving deep proteoform coverage in top-down proteomics critically depends on effective sample pre-fractionation. To address this, we developed PEPPI-MS (Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS) in 2020, leveraging the widely used SDS-PAGE method in biochemistry as a tool for pre-fractionation. PEPPI enables efficient passive extraction of intact proteins from gels, offering a simple, cost-effective, and highly reproducible workflow. Since its introduction, PEPPI has been adopted in various top-down proteomics studies, and more recently, its importance is becoming increasingly more recognized in middle-down proteomics as well. Its potential applications include disease biomarker discovery in top-down/middle-down proteomics although high-throughput processing of large sample cohorts remains a major challenge at present. Automation of the workflow will therefore become essential. In this presentation, we introduce "PEPPI-SP3," our latest workflow combining PEPPI with the magnetic bead-based SP3 method, an established platform for automated sample preparation in bottom-up proteomics, as a promising step forward toward future automation in proteoform-level proteomics.

Speaker: Nobuaki Takemori (Ehime University)

33 Characterization of proteoforms of intact proteins by CE-MS and LC-CE-MS

Electromigrative techniques are powerful tools for the separation of intact proteins and their proteoforms. However, CE-MS is still restricted by the sensitivity and ease-of-use of the interface in conjunction with low injection volumes limiting its application for biological samples. Various solutions will be presented here overcoming these shortcomings.
Initially, the power of CE-MS for the characterization of proteoforms will be presented applying the nanoCEasy interface [1]. Efficient separation of proteins and proteoforms depends strongly on the applied capillary coating. Very recently we developed efficient coatings enabling finetuning the EOF [2] and, thus, increase the separation efficiency for proteins of certain mobility. Results on the application for protein separation from biological samples will be presented.
nanoLC-CE-MS is a promising tool for targeted protein and proteoform analysis in biological samples. Initially a heart-cut nanoLC-CE-MS was setup and the performance regarding improved sensitivity as well as separation of proteoforms was demonstrated [3]. Due to the increased loadability, the nanoLC-CZE-MS setup exhibits a strongly improved increased concentration sensitivity compared to CZE-MS. The combination of high sensitivity and orthogonal selectivity enables the detailed characterisation of intact proteoforms at physiologically relevant concentrations. A novel selective comprehensive online nanoLC-CE-MS configuration will be presented and discussed in the context of targeted proteoform analysis in biological samples using proteoforms of histone as an example.

References
[1] J. Schlecht, A. Stolz, A. Hofmann, L. Gerstung, C. Neusüß, Anal. Chem. 2021, 93, 44, 14593.
[2] L. Dhellemmes, L. Leclercq, H. Frick, A. Höchsmann, N. Schaschke, C. Neusüß, H. Cottet. J. Chrom. A 1720 (2024) 464802.
[3] A. Stolz, C. Neusüß, Analytical and Bioanalytical Chemistry 2022 Vol. 414 No. 5, 1745.

Speaker: Prof. Christian Neusüß (Hochschule Aalen)

34 Impact of sample preparation methods on proteoform identification by top-down proteomics

Numerous workflows have been developed for top-down proteomics (TDP). We systematically investigated the influence of different sample preparation steps on proteoform and protein identifications, including cell lysis, reduction and alkylation, proteoform enrichment, purification, and fractionation [1]. We found that all steps in sample preparation influence the subset of proteoforms identified (e.g., their number, confidence, physicochemical properties, and artificially generated modifications). The various sample preparation strategies resulted in complementary identifications, significantly increasing the proteome coverage. Overall, more than 13,000 proteoforms from more than 2,700 proteins of human Caco-2 cells were identified.
The results presented can serve as suggestions for designing and adapting TDP sample preparation strategies to particular research questions. Moreover, the sampling bias and modifications identified at the intact protein level will also be useful in improving bottom-up proteomics approaches.

[1] Kaulich PT, Jeong K, Kohlbacher O, Tholey A (2024). Influence of different sample preparation approaches on proteoform identification by top-down proteomics. Nat Methods, 21: 2397-2407. doi: 10.1038/s41592-024-02481-6.

Speaker: Andreas Tholey

35 Proteoforms in Tissues – Approaching Their Native Composition with Nano- and Pico-Second Infrared Laser Systems

To analyze tissue molecules accurately, they must be first solubilized. Conventional sampling and homogenization methods disrupt cell compartments, releasing enzymes that can alter proteoforms through proteolysis and modifications of post-translational modifications (PTMs), thereby changing their original composition.
Using nano- or pico-second infrared laser systems (NIRL or PIRL) for tissue sampling minimizes this issue significantly. Their ultrafast sampling and homogenization processes prevent enzymatic activity from altering proteoforms, preserving their native state. Moreover, the gentle, rapid approach reduces fragmentation of proteoforms during sample preparation.
The current NIRL and PIRL systems achieve a spatial resolution of approximately 20 x 20 x 20 µm voxels, approaching single-cell resolution. In summary, NIRL and PIRL-based sampling and homogenization techniques enable a more accurate representation of proteoforms in tissues, bringing analysis closer to their original biological state.

Speaker: Hartmut Schlüter (Institute of Biochemistry)

36 Comparison of RP-LC with CE for histone analysis

Introduction

Histones are heavily and variably decorated by PTMs, thereby affecting their binding to chromosomal regions. Top-down proteomics of histones is advantageous in capturing the PTM combination to obtain epigenetic status. Being rich in lysine residues hampers histone separation by RP chromatography. Capillary electrophoresis (CE) – MS has emerged as powerful alternative for histone analysis. Thus, ZipChip(CE) was benchmarked against nanoRP-LC with a C4 column.

Methods
Calf thymus histone preparation was dissolved in water to 1 mg/ml. For CE 5 ng (5 nl) were injected per analysis and separated in 6 min on a HS CE chip at a field strength of 500V / cm2 in intact antibody BGE buffer. MS and MS/MS spectra were recorded on an orbitrap Ascend set to intact protein mode using instrument templates (<30 kDa intact protein). Protein XML files of Bos Taurus histones were imported into Prosight 4.2/Proteome Discoverer 3.0 for proteoform identification. For LC, 2 µg protein were separated on C4 column in 50 min.

Results and Discussion

The HS chip separated the histones into two major peaks, whereas LC displayed broad elution without baseline separation. The preparation contained histones H1, H2a, H2b, H3, and H4, all of which were identified in the merged MS data (CID, HCD, ETD, EThCD, UVPD). In total, 216 proteoforms and 6291 PrSMs (proteoform spectral matches) were identified at high confidence with CE compared to 569 proteoforms and 35091 PrSMs with RP-LC. PrSMs displayed a most frequent cleavage efficiency of 10%. PrSM count increased from UVPD, EThCD, ETD, HCD to CID. Even though the longer analysis time and higher protein load increased identification, separation performance and speed favored CE. The fast analysis time in combination with very low sample consumption qualify CE as a convenient tool for optimization and data acquisition over a wide range of MS settings.

Speaker: Ansgar Poetsch (Nanchang University)

9:50 AM Coffee Break

New approaches for proteoform analysis

37 Advances in Orbitrap mass spectrometry for top-down analysis

The analysis of intact proteins and protein complexes presents unique challenges to mass spectrometry (MS), prompting a fundamental re-evaluation of principles previously validated for small molecules and peptides. As the initial limitations of fragmentation have largely been overcome in the past two decades—culminating in near-complete freedom of fragmentation in the latest instruments—severe spectral congestion has emerged as a major obstacle, persisting despite advances in modern liquid separation techniques.
Until recently, this issue could only be addressed by high-resolution MS, preferably featuring isotopic resolution and accurate mass. Orbitrap mass spectrometry has proven particularly effective in such analyses, but further progress has been hampered by inherently limited dynamic range for species that overlap in m/z space.
Nowadays, this limitation can be addressed using two distinct approaches: charge reduction and direct charge detection. This presentation explores the practical implementation of both techniques on the latest generation of Orbitrap instruments.
Charge reduction is achieved through high-speed ion–ion reactions, allowing proteins to shed a number of protons and shift to significantly higher and less congested m/z values. By stepping a narrow m/z isolation window in a data-independent manner, this method enables detection of up to ten times more proteoforms—not only for intact proteins, but also for protein complexes. It is also compatible with spectra produced by a broad range of fragmentation techniques.
For direct charge detection, Direct Mass Technology (DMT) allows the charge states of individual ions to be determined in parallel for hundreds to thousands of ions. While typically suited to low-intensity ion beams, DMT is shown to offer intriguing possibilities for top-down analysis as well.
In conclusion, recent rapid advances all aspects of mass spectrometry instrumentation and analysis are shown to open up exciting new possibilities for top-down proteomic analysis.

Speaker: Alexander Makarov (Thermo Fisher Scientific (Bremen) GmbH)

38 Exploring Spatial Top-Down Proteomics

Understanding proteins within their functional contexts, whether in tissue units, cellular neighborhoods, small clusters of cells, or even at the single-cell level, remains a significant scientific challenge that pushes the boundaries of analytical methods. Traditional proteomic approaches largely rely on antibody-based techniques, which limit multiplexing and require prior knowledge of target proteins. While advancements like nanoPOTS-based bottom-up proteomics offer promising tools for analyzing small tissue sections, even single cells, these methods fall short in capturing proteoform-specific information. Proteoforms, representing distinct variations of proteins, are fundamental to cellular roles and functions. To address this gap, we have developed an approach that combines laser capture microdissection (LCM) nanoPOTS with mass spectrometry imaging (MSI), enabling both bottom-up and top-down proteomics. This integrated strategy has been applied across diverse systems, including human, murine, plant, and microbial tissues. For example, in studies of human pancreatic tissue, our methods provided an extensive proteoform landscape, identifying 500-1000 proteoforms and revealing unique variations of endocrine proteins that are often overlooked by conventional methods. MSI further enabled the spatial profiling of hundreds of islets, allowing clustering based on their proteoform signatures. LCM was instrumental for isolating both pooled and individual pancreatic islets, which were then analyzed using nanoPOTS for label-free quantitative top-down proteomics. Additionally, single islets and single cells were dissected to perform comprehensive bottom-up proteomic. These tools are now being applied to investigate chronic pancreatic diseases, T1D progression and therapeutic intervention.

Speaker: Ljiljana Paša-Tolić (Pacific Northwest National Laboratory, Richland, WA 99354, USA)

39 Exploring the effects of isotope depletion on proteins by native mass spectrometry and cryogenic electron microscopy

Isotope depleted protein samples have successfully addressed various challenges in native mass spectrometry (MS), notably by enhancing the signal-to-noise (S/N) ratio- an advantage particularly beneficial for high molecular weight protein analysis. In this study, we explore the broader impact of isotope depletion on reducing sample heterogeneity, and enhancing mass spectral quality in MS, as well as in improving imaging resolution in cryogenic electron microscopy (cryo-EM). Together, these two techniques offer a more detailed visualization of higher-order molecular structure of proteins with high spatial and mass resolutions.
We successfully expressed and purified test proteins of varied MW, in isotope depleted media. Native mass spectra of these samples showed a distinctive shift towards monoisotopic peaks and a reduction in mass window as compared to the proteins grown in normal media, confirming effective isotope depletion. The improved S/N ratio also enhanced sequence coverage in top-down proteomic analysis in isotope depleted proteins compared to the normal ones. The study extends to cryo-EM to assess the potential improvements in imaging resolution and any structural alterations induced by isotope depletion.

Speaker: Anjusha Mathew

40 Discovering the ‘negative’ side of the proteomic landscape with top-down mass spectrometry

Conventional mass spectrometry-based proteomics use positive polarity and provide a wealth of qualitative and quantitative information; however, these methods may not capture the true complexity of the proteome. Our work aims at alleviating this issue by resorting to ionization in negative polarity to account for the acidic portion of the intact proteome that preferentially ionizes as anions. Negative polarity bottom-up strategies have already shown utility in boosting coverage of acidic peptides and our objective was to extend this strategy to the top-down (i.e., intact protein) domain to discover acidic species at the proteoform level, as well. Standard polypeptides with molecular weights between 1-48 kDa were interrogated by HCD, CID and UVPD using both polarities, whereby cleavage propensities and the effect of charge density were determined. A unique feature of negative mode HCD and CID is their ability to cleave disulfide-linkages more readily than in positive polarity. While cleaving disulfides is appealing, using negative mode HCD and CID comes at the cost of producing a high ratio (>50% of the total ion current) of neutral losses, including series of consecutive ammonia, water and CO2 losses. These additional peaks make spectral interpretation challenging and negatively impact the p-score of proteoform anions. Measurements conducted on the LC timescale for a standard protein mixture (Pierce standard) using high-pH separation on a polymeric resin seem promising and we are working on introducing new bioinformatic tools for conducting database searches for negative mode data.

Speaker: Cynthia Nagy (University of Oklahoma)

41 Glycoproteomics Based on Deep Learning and Data Independent Acquisition

Large-scale profiling of intact glycopeptides is critical but challenging in glycoproteomics. In 2021, we propose GproDIA [1], a framework for the proteome-wide characterization of intact glycopeptides from DIA data with comprehensive statistical control by a 2-dimentional false discovery rate approach and a glycoform inference algorithm, enabling accurate identification of intact glycopeptides using wide isolation windows. We benchmark our method for N-glycopeptide profiling on DIA data of yeast and human serum samples, demonstrating that DIA with GproDIA outperforms the data-dependent acquisition-based methods for glycoproteomics in terms of capacity and data completeness of identification, as well as accuracy and precision of quantification.
In 2024, we further present DeepGP [2], a hybrid deep learning framework based on Transformer and graph neural network (GNN), for the prediction of MS/MS spectra and retention time of glycopeptides. Testing on multiple biological datasets, we demonstrate that DeepGP can predict MS/MS spectra and retention time of glycopeptides closely aligning with the experimental results. Comprehensive benchmarking of DeepGP on synthetic and biological datasets validates its effectiveness in distinguishing similar glycans. Remarkably, DeepGP can differentiate isomeric glycopeptides using MS/MS spectra without diagnostic ions.
More recently, we present a method using the ZenoTOF instrument with optimized fragmentation for intact glycopeptide identification and demonstrate its ability to analyze large-cohort glycoproteomes[3]. From 124 clinical serum samples of breast cancer, non-cancerous diseases, and non-disease controls, a total of 6901 unique site-specific glycans on 807 glycosites of proteins were detected. Much more differences of glycoproteome were observed in breast diseases than the proteome. By employing machine learning, 15 site-specific glycans were deter-mined as potential glyco-signatures in detecting breast cancer.
[1] Nature Communications, 2021, 12, 6073
[2] Nature Machine Intelligence, 2024, 6, 950-961
[3] Analytical Chemistry, 2025, 97, 114-121

Speaker: Liang Qiao (Fudan University)

42 High resolving power meets proton transfer charge reduction: unlocking new depths in intact protein characterization

Although the characterization of intact proteins remains a demanding endeavour – particularly as protein size increases - ongoing technological/bioinformatic advancements are steadily enhancing the capabilities of top-down mass spectrometry (TDMS) for deep sequencing. Different ion activation techniques – such as electron transfer dissociation (ETD), electron transfer higher-energy collisional dissociation (EThcD), and ultraviolet photodissociation (UVPD) available on the Thermo Scientific Orbitrap Tribrid MS platform - can provide complementary fragmentation patterns, and when combined, offer elevated sequence coverage. Despite the advancements in fragmentation techniques, data interpretation remains challenging due to extensive overlapping of ion signals, which compromises product ion matching. Herein, we present strategies for resolving spectral ambiguities, implemented on the Orbitrap Ascend BioPharma MS.
Proteins in the 8-46 kDa Mw range were interrogated with higher-energy collisional dissociation (HCD), ETD, EThcD, and UVPD. Data collection was carried out using a range of resolving powers (RP). A gradual increase in coverage was observed moving from an RP of 60,000 towards 480,000 in all cases - for enolase reaching 53% from the initial 14% yielded by lower resolution. However, high RP alone could not effectively resolve spectral congestion due to fragment ions signals being restricted to m/z < 2000. Additional increase in coverage (~8-15%) could only be achieved with proton transfer charge reduction (PTCR), due to its ability to distribute the product ion population over a broader m/z range (up to m/z 8000). The PTCR MS3 workflow yielded a coverage of 98% for carbonic anhydrase (~29 kDa) when results from all tested fragmentation techniques were combined.
To evaluate the approach under chromatographic time constraints, we applied the workflow for the middle-down analysis of mAb and antibody-drug conjugate (ADC) ~25 kDa subunits. PTCR MS3 enabled not only increased coverage (87% for Fd’ subunit) but also the unambiguous localization of payload conjugation sites for the ADC.

Speaker: Rafael D. Melani (Thermo Fisher Scientific, San Jose)

12:00 PM Lunch Seminar (Bruker Daltonics)

Poster Session 2

Biomedical Applications

43 Top-Down Proteomics of the Heart: Decoding Cardiac Proteoforms for Precision Medicine

Proteoforms - encompassing the diverse protein products arising from alternative splice isoforms, genetic variations, and posttranslational modifications (PTMs) originating from a single gene - are fundamental drivers in biology. Top-down mass spectrometry (MS)-based proteomics (TDP), analyzing whole proteins without digestion, offers a comprehensive perspective of proteoforms, which is invaluable in deciphering proteoform function, uncovering disease mechanisms, and advancing precision medicine. We have been developing novel technologies to address the challenges in top-down proteomics in a multi-pronged approach including new cleavable surfactants for protein solubilization, new strategies for multi-dimensional chromatography separation of proteins, novel nanomaterials for enrichment of low-abundance proteins. In this presentation, I will highlight the application of TDP to enable proteoform-resolved analysis of cardiac proteins directly from human heart tissues and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Notably, we have identified altered cardiac proteoforms associated with contractile dysfunction. Through case studies in hypertrophic, ischemic, and dilated cardiomyopathy, we demonstrate how TDP uncovers disease mechanisms, reveals novel biomarkers, and informs therapeutic strategies. By mapping the proteoform landscape of the human heart, top-down proteomics has the potential to transform cardiovascular research and enable more precise, individualized interventions.

Speaker: Ying Ge (University of Wisconsin-Madison)

44 Dissecting the Proteoform Landscape of Prostate-Specific Antigen: Intact, Bottom-Up, and Glycomic Perspectives

Early detection of prostate cancer (PCa) using prostate-specific antigen (PSA) serum levels suffers from poor specificity and sensitivity, frequently causing unnecessary treatment or missed diagnoses. To overcome this, detailed characterization of PSA proteoforms, especially regarding glycosylation, is essential. Here, we employed an integrated analytical strategy using capillary electrophoresis coupled with mass spectrometry (CE-MS), progressively scaling from micro-level glycan details to macro-level intact protein analysis.

At the micro-level, released N-glycan profiling provided validation of glycosylation patterns identified by peptide-level (bottom-up) and intact analyses. The bottom-up approach offered detailed differentiation of glycopeptides, particularly distinguishing α2,3- from α2,6-linked sialic acid isomers through differences in electrophoretic mobility correlated with subtle pKa variations (relative pKa difference: 3.4×10⁻²). This high-resolution separation uniquely revealed precise structural features, including the first identification of ketodeoxynononic acid (Kdn) on PSA glycans derived from seminal plasma and urine.

Moving to the macro-level, intact protein analysis delivered a global view of PSA proteoforms, capturing six proteolytic cleavage variants alongside diverse glycosylation states—including tri-, di-, mono-, and non-sialylated glycans—and, for in one of the patients urinary samples, uncovered a second glycosylation site resulting from genetic mutation.

Together, these complementary methodologies overcome individual analytical limitations, offering an extensive characterization of PSA proteoforms and their glycomic complexity. Future research will evaluate whether the proteoform diversity can be utilized to enhance discrimination between aggressive PCa, indolent PCa, and benign prostate hyperplasia. Additionally, further studies will investigate glycomic variations in plasma, preserve native PSA molecular complexes, and assess the clinical relevance of proteoform diversity in relation to disease severity and progression in larger patient cohorts.

Speaker: Guinevere Lageveen-Kammeijer (University of Groningen)

45 Spatial Phosphoproteomic Profiling Reveals Regional Functional Heterogeneity in the Murine Heart

Phosphorylation mediated signaling is fundamental to cardiac function. However, the dynamic signaling patterns across different spatial regions of the heart have been inadequately explored. This is mainly because of the technical difficulties in analyzing tiny tissue samples with the required depth and sensitivity. To tackle these challenges, we developed an optimized TiO₂ based micropipette tip method for in-depth phosphoproteomics. This methodology showcases outstanding sensitivity, identifying 12,173 class I phosphosites from 10 µg HeLa peptides, and also offers high reproducibility.
We then utilized this advanced technique to study spatially defined regions of the mouse heart. Through laser-capture microdissection, we isolated seven specific anatomical areas: the left atrium (LA), right atrium (RA), left ventricle (LV), right ventricle (RV), interventricular septum (IVS), apex (APEX), and aortic valve (AV). For each region, we were able to quantify 1,000-2,000 phosphosites. Principal component analysis revealed distinct phosphoproteomic signatures that cluster according to anatomical positions, providing higher resolution differentiation than proteomic profiling. Functional enrichment analysis further unveiled region-specific phosphorylation patterns. The APEX and ventricular regions were characterized by phosphorylation signatures associated with the contractile machinery. In the AV tissue, proteins related to cell junctions and polarity were significantly enriched. From a metabolic perspective, the LV exhibited phosphorylation patterns closely tied to energy metabolism, while the LA showed enrichment in RNA processing pathways. Phosphoproteins in the RA were predominantly involved in cellular component biogenesis and chromatin organization.
This spatially resolved atlas establishes a molecular foundation for investigating region-specific cardiac pathologies and advances understanding of post-translational cardiac heterogeneity.

Speaker: Ling Lin (Fudan University)

46 Spatially Resolved Proteoform Mapping in Alzheimer’s Disease Brain Tissues

Understanding spatial heterogeneity of proteoforms has great potential to unravel physiological and disease mechanisms. However, conventional proteomics often lacks spatial information or focuses on the protein-coding gene level, while existing methods for spatial proteoform analysis suffer from low throughput or limited coverage. These gaps hinder the exploration of spatially resolved proteoform-function relationships
In this work, we developed high-throughput proteoform imaging (HTPi), an integrative workflow combining matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) with deep annotation via region-specific top-down proteomics, using a custom-designed narrow-bore monolithic column to enhance sensitivity. HTPi achieved proteoform visualization at 20–100 μm spatial resolution and annotated 366 proteoform images in mouse brain tissues, revealing distributions of individual proteoforms across different brain regions and distinct spatial patterns of proteoforms from a single gene (e.g., six Pcp4 proteoforms).
Applied to 5×FAD mice, HTPi was used to explore proteoform perturbations in the hippocampus, cortex, thalamus, and striatum. Notably, HTPi uncovered Aβ proteoforms (1–38, 1–40, 1–42) localized to subiculum plaques and identified 14 differential proteoforms, including truncated Ubb and mitochondrial Ndufv3. The co-localization of truncated Ubb proteoforms with Aβ plaques suggested a link between Aβ accumulation and ubiquitin-proteasome dysfunction. These results highlighted HTPi’s ability to resolve proteoform-level spatial dynamics in Alzheimer’s disease pathogenesis.
By bridging high-throughput MALDI MSI with region-specific top-down proteomics, HTPi advances spatial proteomics, offering insights into molecular mechanisms and potential biomarkers for neurodegenerative diseases. Future applications may expand to 3D brain-wide proteoform mapping and clinical translation for disease diagnosis.

Speaker: Yue Sun (Dalian Institute of Chemical Physics, Chinese Academy of Sciences)

47 Top-down Proteomics Deciphers Cardiac Proteoform Landscape in Phospholamban R14del Cardiomyopathy for Precision Medicine

Phospholamban (PLN) is a transmembrane protein that regulates cardiomyocyte calcium handling and contraction. PLN function is dynamically regulated by post-translational modifications (PTMs), most notably through phosphorylation. A pathogenic deletion of arginine-14 (PLN-R14del), is associated with dilated cardiomyopathy (DCM), and defined by a high mortality rate with minimal treatment options. Moreover, the molecular mechanism of pathogenesis remains unclear, as some R14del carriers are asymptomatic. We use mass spectrometry (MS)-based top-down proteomics to investigate proteoform alterations in two systems: 1) human cardiac tissue from healthy donors and late-stage DCM patients (R14del carriers and noncarriers); 2) human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from symptomatic and asymptomatic patients. Proteins were extracted from cryopulverized tissue or hiPSC-CMs using a two-stage extraction. Cytosolic proteins are depleted, while PLN and additional cardiac proteins were extracted with Azo (MS-compatible surfactant). Proteins were buffer exchanged and analyzed with reverse phase liquid chromatography coupled to tandem MS. Characterization of the proteoform landscape in R14del patients revealed multiple PLN proteoforms. Notably, we observed a significant decrease in the total phosphorylation levels of R14del samples compared to both donor samples and DCM patients without the mutation, providing a novel insight into the endogenous phosphorylation potential of PLN-R14del. Additionally, we observed dysregulation of phosphorylation in key sarcomere/Z-disk proteins, further associating contractile dysfunction with proteoform alterations. In the hiPSC-CM model, preliminary analysis revealed that the asymptomatic patient line contained PLN phosphorylation levels comparable to the isogenic control line; importantly, there was a significant increase in phosphorylation in both when compared to the symptomatic carrier. These results suggest that proteoforms may factor into variable disease expressivity and that our method is capable of delineating distinct phenotypes between patients that share the same mutation, which can aid in the pursuit of therapeutic strategies for precision medicine and bridge the gap between genotype and phenotype.

Speaker: Holden Rogers (University of Wisconsin-Madison)

48 MALDI MS-Based Rapid Antimicrobial Susceptibility Prediction

Bacterial infections are among the diseases with high morbidity and mortality rates worldwide, posing a significant threat to global public health. There is an urgent need to develop precise and rapid diagnostic methods for bacterial infections to enable personalized medication and treatment for infected patients, promptly save lives, and reduce the spread of antimicrobial resistance. Bacterial infection diagnosis encompasses two key aspects: bacterial identification and antibiotic susceptibility testing (AST). Current clinical methods for bacterial identification and AST are limited by the time-consuming process of bacterial culture. Bacterial identification is typically performed using MALDI-TOF MS, while AST is conducted with automated biochemical analyzers, requiring an additional step of proliferation testing under antibiotic stimulation, resulting in a delay of 6~24 hours compared to bacterial identification.
To accelerate antibiotic susceptibility testing (AST) and reduce costs, we have developed two rapid AST methods based on MALDI-TOF MS. The first method detects deuterium incorporation into newly synthesized proteins under antibiotic stimulation, allowing for monitoring of protein synthesis and using machine learning to predict bacterial susceptibility. This approach introduces a series of discriminative features, resulting from mass shifts induced by deuterium incorporation, which significantly enhances the performance of machine learning models, especially on small datasets. Additionally, when transferring training results from public datasets to smaller datasets, this method improves the accuracy of antibiotic susceptibility predictions. The second method monitors changes in bacterial metabolites under short-term antibiotic stimulation and, when combined with machine learning, also predicts antimicrobial susceptibility. Both methods reduce AST time to just 0.5 to 1 hour after bacterial identification by MALDI MS. Furthermore, these approaches integrate both AST and bacterial identification into a single mass spectrometer, facilitating faster diagnoses, reducing equipment and labor costs, and demonstrating great potential for broader clinical applications of mass spectrometry.

Speaker: Dr Jia Yi (Minhang Hospital, Fudan University)

3:25 PM Coffee Break

Biopharmaceutical & Therapeutic Proteins

49 Deciphering Biotherapeutic Biotransformations with Top-Down Mass Spectrometry

Today, within the R&D pipelines of pharmaceutical companies, monoclonal antibodies are gradually being replaced by new-generation biotherapeutics, including engineered hybrid and multispecific constructs. While these innovative formats hold great therapeutic promise, their structural complexity often leads to unexpected in vivo instabilities that may compromise efficacy or alter pharmacokinetics. Understanding their metabolic fate is therefore crucial to guide rational design and ensure functional performance.

However, current analytical workflows—originally designed for small molecules or peptide-level proteomics—struggle to capture the full heterogeneity of these large, hybrid biomolecules. Proteolytic digestion breaks apart structurally distinct proteoforms into overlapping peptide mixtures, leading to a loss of connectivity and incomplete characterization.

To overcome these limitations, we have developed and applied complementary middle-down and top-down mass spectrometry strategies capable of analyzing intact proteins and large subunits (25–100 kDa). These approaches were implemented on the timsOmni multimodal platform (Bruker) and the Orbitrap Eclipse Tribrid system (ThermoFisher), allowing high-resolution analysis with advanced dissociation methods (ECD, EID, CID, ETD, HCD).

We demonstrate that these techniques can detect and partially sequence low-abundance mAb proteoforms directly from in vivo plasma samples following administration in mice. A key part of this work involved the automated immunoenrichment of biotherapeutic metabolites from complex biological matrices, enabling their isolation and characterization despite low concentrations and high background.

Together, these innovative analytical workflows provide new insight into biotherapeutic metabolism, including the detection of truncated forms resulting from rapid in vivo cleavage—such as the loss of target-binding domains—which directly impacts the therapeutic mechanism of action. These results highlight the importance of proteoform-level analysis for next-generation biologics and open the path toward more robust, predictive tools in biotherapeutic development.

Speaker: Julia CHAMOT-ROOKE (INSTITUT PASTEUR - CNRS)

50 Functional and structural characterization of antibodies by native-mode affinity separation-, middle-up, and top-down mass spectrometry

Antibodies show tremendous structural diversity shaping their biological functions in immunity and immune pathologies. While IgG1 Fc domains have been extensively characterized by MS approaches, more complex antibody structures (including Fab glycosylated IgGs or isotypes such as IgA or IgM) are less well understood. This presentation showcases the integration of miniaturized bottom-up, middle-up and intact mode workflows for dissecting the Fab and Fc hetereogeneity of endogenous antibodies, with a particular focus on defining glycoform profiles. By employing specific capturing approaches in combination with selective hinge-region cleavage strategies, specific antibody populations and fragments are isolated and characterized revealing intrinsic differences. Native-mode affinity-capillary electrophoresis with mass spectrometry allows to assess how antibody proteoforms including Fc and Fab glycosylation define Fc-receptor interactions. This work provides insights into molecular details of antibody functionalities in health and disease.

We applied the middle-up approach to characterize IgG Fc portions of anti‐citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) from both synovial fluid and plasma samples, and compared them to the proteoform profiles of total or bulk IgG from the same samples. We observed differences in isotype and allotype usage as well as Fc glycosylation between the different antibody populations. Remarkably, both IgG1 agalactosylation and IgG4 subclass usage appeared to associate with disease activity as well as erythrocyte sedimentation rate, providing indications of a possible contribution of these antibody variants to RA etiology.

References

Blöchl et al. 2025 Fc Proteoforms of ACPA IgG Discriminate Autoimmune Responses in Plasma and Synovial Fluid of Rheumatoid Arthritis Patients and Associate with Disease Activity. Advanced Sciences. e2408769. doi: 10.1002/advs.202408769.

Gstöttner et al. 2024 Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding. Front Immunol. 15:1347871. doi:10.3389/fimmu.2024.1347871.

Speaker: Manfred Wuhrer (Leiden University Medical Center)

51 AiDA Accelerates Top-Down and Middle-Down MS Data Analysis Across Multiple Antibody Variants

Mass spectrometry (MS) is essential for characterizing biotherapeutics, with Top-Down (TD) and Middle-Down (MD) approaches offering faster alternatives to peptide mapping. Despite achieving high sequence coverage using advanced fragmentation techniques like HCD, ETD, and UVPD, traditional data analysis presents challenges. These include time-consuming fragmentation map creation, difficulty localizing post-translational modifications (PTMs) in non-fragmented regions, and missed diagnostic ions due to monoisotopic peak detection errors in complex spectra.
To overcome these issues, the All ion Differential Analysis (AiDA) method was developed for online antibody variant characterization. (1) AiDA enables rapid identification of diagnostic spectral differences across multiple MS spectra before fragment assignment, significantly accelerating data analysis. It introduces a quantitative layer to TD and MD workflows by analyzing preferential fragmentation patterns, particularly near aspartic and iso-aspartic acid residues, to localize PTMs with statistical confidence.(1, 2) AiDA also helps detect interactions between neighboring residues and has proven effective in characterizing deamidation, sequence variant and sequence positional isomers of oxidation and iso-aspartic acids in antibodies. Finally, AiDA supports multi-level validation of internal fragments N-terminal to Proline which can be used to increase MD sequence coverage by up to 34% and access otherwise non-fragmented regions.(1) Both 1D and 2D AiDA applications will be discussed.

(1) Griaud F, Denefeld B, Kao-Scharf CY, Dayer J, Lang M, Chen JY, Berg M. Anal Chem. 2019 Jul 16;91(14):8845-8852.
(2) Denefeld B, Hajduk J, Cerar J, Rondeau JM, Dayer J, Lang M, Kern W, Griaud F. J Am Soc Mass Spectrom. 2025 May 7;36(5):969-979.

Speaker: Francois Griaud (Novartis Pharma AG)

52 Enhanced usage of top-down data for de novo sequencing of antibodies

The Twister algorithm (Vyatkina et al., 2015, 2016, 2017), initially intended for de novo sequencing of antibodies from top-down MS/MS data supported with high-resolution bottom-up MS/MS spectra, is being developed further – currently aiming, at particular, at taking the maximum profit from internal fragment ions. In this talk, we will present the latest version of the Twister algorithm, along with the most recent results obtained.

Speaker: Kira Vyatkina (Sechenov University)

53 Mass spectrometric ITEM-FOUR analysis reveals coding single nucleotide polymorphisms in human cardiac troponin T that evade detection by sandwich ELISAs which use monoclonal antibodies M7 and M11.7 from the Elecsys Troponin T® assay

Immunoassays for cardiac troponin, such as the Elecsys® hs-TnT, have become the gold standard for myocardial infarction diagnostics. While various protein/chemical factors affecting the troponin complex and, thus, its diagnostic accuracy have been investigated the role of coding single nucleotide polymorphisms remains underexplored. To evaluate potential cSNP-induced interference with antibody binding in the Elecsys® hs-TnT immunoassay, we applied ITEM-FOUR, a mass spectrometry-based method that quantifies changes in antibody binding upon amino acid substitutions in epitope pep-tides. Candidate cSNPs were selected from the dbSNP database and were mapped to human cardiac troponin T by molecular modeling. Consuming micromolar antibody concentrations and microliter sample volumes, two wild-type and 17 cSNP-derived variant epitope peptides—six for monoclonal antibody M7 and eleven for monoclonal antibody M11.7—were investigated to reveal the binding motifs 'V131-K134-E138-A142' for M7 and 'E146-I150-R154-E157' for M11.7. Loss of binding to M11.7 was observed for substitutions Q148R (rs730880232), R154W (rs483352832), and R154Q (rs745632066), whereas the E138K (rs730881100) exchange disrupted binding of M7. Except for cSNP Q148R they are associated with cardiomyopathies, placing affected individuals at risk for both, underlying heart disease and false-negative hs-TnT assay results in case of myocardial infarction. Our results highlight the need to account for cSNP-related interferences in antibody-based diagnostics. ITEM-FOUR offers a powerful approach for tackling this challenge, fostering next-generation assay development.

Speaker: Michael Glocker

5:20 PM Poster Awards and Farewell

Fri, August 29

Bruker Factory Tour

Short Course 2: Dissecting the impact of deconvolution on top-down identification

9:00 AM Assessing the degree of variation among deconvolution engines (David Tabb, Tom Müller, Kyowon Jeong)

Use of TopFD / FLASHDeconv,
deconv results in Jupyter

10:30 AM Coffee Break

11:00 AM Detecting deconvolution impact on identifications (David Tabb, Tom Müller, Kyowon Jeong)

Use of TopPIC Identifications in Jupyter

1:00 PM Lunch