Proteases are common components of extracellular vesicles (EVs), yet the extent and functional relevance of ongoing proteolytic activity on EV surfaces remain largely unexplored. Such activity could significantly influence EV function and identity, with likely implications for EV-mediated signalling, recipient cell targeting, cargo delivery, and even translational applications ranging from biomarker discovery to therapeutic approaches. Here, we investigated the impact of sustained proteolysis on the composition of brain cell-derived EVs, focusing on A Disintegrin And Metalloprotease 10 (ADAM10), a key sheddase for signalling and adhesion proteins involved in neuronal and synaptic processes. Using primary rat cortical cultures, we found that numerous known ADAM10 substrates are part of small EVs (sEVs), and that their associated functions overlap with major sEV-mediated roles such as nervous system development, cell adhesion, and neurite outgrowth. Applying N-terminal proteomics to monitor sEV-derived cleavage fragments over time, we identified novel substrate candidates and demonstrated that sEV-associated ADAM10 activity remodels surface proteins involved in EV-cell interactions while generating soluble factors implicated in neuronal development. These findings suggest a previously unrecognised role for ADAM10 as a modulator of sEV composition and potentially cell-targeting specificity in the brain and position EVs as dynamic platforms for proteolytic processing 'on the move'.

Small open reading frame (sORF)-encoded proteins, with less than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in classical methodologies. For the mesophilic archaeal model system Methanosarcina mazei, a high number of previously unannotated sORFs have recently been identified. However, the physiological role of most of the respective small proteins remains unknown. Here, we report on the functional characterization of the small ORF16-encoded small protein MtrR (49 amino acids). We demonstrate that MtrR forms oligomers localized at the cytoplasmic membrane. There, it interacts with the tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex of energy metabolism, and impacts its activity. In vitro interaction and in vivo copurification assays showed MtrR interaction with the Mtr complex, which was further validated by microscale thermophoresis analysis demonstrating a specific interaction with the MtrA subunit. Analyzing growth under varying molecular hydrogen (H2) availability demonstrated that the mtrR deletion mutant showed significantly impaired growth in the presence of H2, independent of the carbon source. Further, we observed induction of mtrR transcription in the presence of H2. Consequently, we propose that MtrR fine-tunes the activity of the Mtr complex in response to fluctuating H2 availabilities, allowing adaptation of the energy metabolism to changing environmental H2 conditions.

Meprin α and meprin β are zinc metalloproteases that are strongly expressed in intestinal and renal tissues and are expressed as homo- and heterodimers. In the kidney and intestine, they are involved in extracellular matrix assembly and modulation of inflammatory responses. However, meprin β has recently attracted attention because it generates Alzheimer's Disease (AD)-specific Aβ peptides and cleaves brevican, a major component of the perineuronal nets (PNNs) in the brain. PNNs stabilize synapses, thereby regulating plasticity and memory formation. Brevican cleavage correlated with impaired spatial memory formation and impaired CA1 long-term potentiation (LTP) in meprin β transgenic mice. Furthermore, numerous studies have shown the dysregulation of PNN components in AD. Still, the physiological and pathological functions of proteolytic PNN remodeling remain elusive. This study identified an essential role of meprin α in brevican cleavage. It enhanced meprin β's catalytic activity on brevican in co-expression. Moreover, an N-terminomics analysis identified novel meprin β substrates, neurocan, and receptor-type tyrosine-protein phosphatase zeta (RPTPζ) in the brain. Both are key components of PNNs. RPTPζ cleavage by meprin α and meprin β was confirmed in vitro. To assess the functional impact of meprin-mediated proteolysis on the brain extracellular matrix, PNNs and synaptic organization were investigated in vivo using immunofluorescence and electron microscopy. Meprin-mediated proteolysis disrupted PNN structure and decreased synapse density in the hippocampal CA1 region of meprin β transgenic mice. This identifies meprin-dependent PNN remodeling as a novel mechanism contributing to synaptic dysfunction.

Lysozymes are an essential part of immunity and nutrition in metazoans, degrading bacterial cell walls via the hydrolysis of peptidoglycan. Although various lysozymes have been reported for higher animals, the origin of animal lysozymes remains elusive as they seem to be lacking in all early branching phyla. In this study, we investigated a putative goose-type lysozyme (PLys, glycoside hydrolase family 23, GH23) of the placozoan Trichoplax sp. H2. We show that PLys is highly active and primarily produced by cells of the placozoan ventral epithelium. PLys contains a non-conserved cysteine-rich domain N-terminal of the GH23 lysozyme domain, which stabilizes the protein and is truncated during maturation. Using a pH-sensitive fluorescence reporter, we show that Trichoplax sp. H2 acidifies its temporary feeding grooves pulsatively during digestive events close to the optimum pH for PLys activity. To elucidate the evolutionary origin of the metazoan GH23 lysozyme family, we applied structure-based phylogenetics to show that the metazoan g-type GH23 lysozymes originated from a horizontal gene transfer event from bacteria to an early pre-bilaterian ancestor. GH23 lysozymes have then been retained and expanded in many phyla acting as first animal lysozyme and a key component in the antibacterial arsenal since early animal evolution.

Top-down mass spectrometry provides a powerful approach for analyzing and quantifying intact proteoforms, i.e., the distinct molecular forms of proteins. Isobaric labeling-based quantification strategies offer the advantages of multiplexing and increased analytical depth. However, a major challenge remains the quantification of proteoforms when their precursor signals overlap, leading to mixed reporter ion intensities. In this proof-of-concept study, we employed proton transfer charge reduction (PTCR) at the MS2 level to resolve overlapping precursor signals, allowing selective isolation of individual proteoforms and subsequently, their accurate reporter ion quantification at the MS3 level. Using direct infusion mass spectrometry of model proteins labeled with cysteine-directed tandem mass tags, we demonstrate that this approach enables accurate, interference-free reporter ion-based quantification in the presence of overlapping proteoforms and spectrally congested backgrounds. This work highlights PTCR as a versatile gas-phase separation strategy to enhance the quantitative capabilities of labeling-based top-down mass spectrometry, offering a path toward precise, proteoform-resolved quantification across diverse experimental approaches, such as large-scale top-down proteomics.

Abstract Understanding proteoforms, i.e., the various molecular forms in which proteins can exist, is important for deciphering biological processes and diseases. While capillary zone electrophoresis (CZE) proved advantageous for proteoform separation, limited sample loading capabilities restrict its application. Here, we present a novel comprehensive two-dimensional nanoLCxCZE-MS platform for deep top-down proteomics (TDP). The 2D platform is highly automated, enabling robust performance and the possibility to perform proteoform quantitation as demonstrated by isobaric labeling experiments. The high orthogonality of reversed-phase LC and CZE leads to a peak capacity of 2200, leading to an increase in the number of identified proteoforms in a human Caucasian colon adenocarcinoma cell lysate sample by a factor of 3 compared to nanoLC-MS. Furthermore, CZE mobilities enable the attribution of many more proteoforms to a certain proteoform family on the MS1-level. Overall, the flexible platform enables highly efficient separation of intact proteoforms combined with sensitive MS-based TDP workflows, both for untargeted and targeted analysis of complex biological samples. Graphical Abstract We report a robust and automated comprehensive nanoLCxCZE-MS platform for top–down proteomics. In addition to large volume sample injection and separation by hydrophobicity in the nanoLC, the orthogonal separation by CZE in the second dimension leads to a strong increase in peak capacity and, thus, in the number of identified proteoforms. CZE mobilities also enable the attribution of many more proteoforms to a proteoform family on the MS1-level.

Seafood, including crustaceans, provides a high-quality animal-origin protein source with globally increasing consumption rates. However, international seafood trade is frequently confronted with issues like commercial fraud and safety risks for human health, partly due to incorrect species identification of aquatic food products. To distinguish between crustacean species through the identification of species-specific peptides, an untargeted liquid chromatography low-resolution mass spectrometry (LC-LRMS) method was developed and validated. Programmed algorithms were applied to identify marker candidates from peptide profiles. A targeted multiple reaction monitoring (MRM) method was established to verify the suitability of selected candidates as crustacean biomarkers related to the used dataset. An additional random forest model was built to determine unknown crustacean species based on an LC-LRMS raw data training set. In total, 49 out of 150 selected peptides were identified as species-specific biomarkers based on the monitored dataset and those are able to differentiate 14 crustacean species. To further evaluate specificity, additional commercial samples from several crustacean, mussel, insect and fish species were tested. De novo sequencing of LC-LRMS and comparing high-resolution mass spectrometry (HRMS) data mostly showed similar results concerning the proposed amino acid sequence and average local confidence score. Selected peptide markers were synthesized and experimentally confirmed. The random forest model exemplarily demonstrated the correct identification in an unseen test dataset based on plurality vote. The results show the feasibility of solving authenticity questions, e.g. to identify unknown crustacean species, by applying alternative procedures without using HRMS instruments, requiring a higher effort. At the same time, the results prove that certain limitations cannot be overcome using LRMS devices.

The emergence of Alzheimer's disease (AD) pathology has been the focus of multiple hypotheses, with amyloid β (Aβ) playing a central role due to its presence in both familial and sporadic AD. Therefore, a crucial aspect of AD research is understanding the generation of different Aβ species. Aβ peptides result from the proteolytic processing of Amyloid Precursor Protein (APP) by β- and γ-secretases, with BACE1 being the most prominent β-secretase. However, BACE1-overexpressing mouse models exhibit disadvantages, making them limited for AD research. Importantly, N-terminally truncated Aβ species, which constitute up to 70 % of Aβ in AD brains, are not generated by BACE1. In recent years, alternative proteases capable of cleaving APP have been identified, bridging the gap between N-terminally truncated Aβ species and BACE1-derived Aβ. Among these novel players, the metalloprotease meprin β has emerged as a risk factor in AD pathology, generating both N-terminally truncated and full-length Aβ species. Our primary objective was to develop a mouse model that more accurately resembles the pathology of AD beyond BACE1-overexpressing models, while simultaneously confirming APP cleavage of meprin β in the hippocampus and cerebral cortex. Overexpression of meprin β led to a marked increase in soluble Aβ levels, particularly in the hippocampus, indicating a higher vulnerability or elevated meprin β activity in this region compared to the cerebral cortex. Notably, this biochemical change occurred without any observable behavioral deficits, suggesting a region-specific role of meprin β in AD pathology that may extend beyond immediate functional impairment.

The metalloprotease meprin β is known for its multifunctional involvement in various physiological processes throughout the body including the brain. However, its broader functions within the brain besides amyloid β generation remain largely unexplored. To investigate this, we utilized a mouse model overexpressing meprin β in neurons within the cortex and hippocampus, regions crucial for learning and memory. Behavioral assessments, employing the Morris' Water Maze paradigm test, revealed impaired cognitive functions in animals overexpressing meprin β. Furthermore, electrophysiological recordings in hippocampal slices using multielectrode arrays showed an impaired long-term potentiation (LTP) in meprin β-overexpressing mice compared to wild-type counterparts. Intriguingly, concomitant with the LTP impairment, we observed an increased neuronal excitability. These findings underline the complicated interplay between meprin β abundance and behavioral manifestations, suggesting a broader impact on neural circuit dynamics. To elucidate the molecular mechanisms underlying these observed deficits, western blotting analyses were conducted to address the expression of glutamatergic receptors. Neither the expression of the N-methyl-D-aspartate (NMDA) nor the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor showed variation relative to each other. The application of N-terminomics identified brevican as a proteolytic substrate of meprin β and thus a potential key mediator linking meprin β overexpression to the observed effects. Previous studies have reported that brevican knockout in animal models influences learning and memory. Our data demonstrate that meprin β modulates brevican expression, likely contributing to the effects we have observed in our mouse model. These results shed light on the broader functional significance of meprin β in neurological processes.

Background: A metabolic switch favoring glycolysis over aerobic oxidative phosphorylation, namely the 'Warburg effect', represents a hallmark of cancer cells. Hexokinases (HK) catalyze the first step of glycolysis, thereby regulating its rate. Dysregulated HKDC1 (HK domain containing 1) expression has been associated with various cancer types and blocking HKDC1 prevents disease progression for hepatic carcinoma T cell lymphoma and lung adenocarcinoma, but its implication for colorectal cancer (CRC) remained unknown. Here, we functionally investigated the role of HKDC1 for intestinal carcinogenesis. Methods: First, we analyzed HKDC1 expression in the intestinal mucosa of healthy controls (HC) and CRC patients and in different tumor tissues using transcriptomic data from publicly available databases. We then generated HKDC1-deficient human and murine colonic epithelial cell lines as well as intestinal organoids and profiled their phenotypic functions. Next, we screened for proteins interacting with HKDC1 by immunoprecipitation. Finally, we generated tumor-bearing ApcMin/+ mice with a conditional deletion of HKDC1 in intestinal epithelial cells and also performed a xenograft mouse model to test the role of HKDC1 for intestinal carcinogenesis in vivo. Results: HKDC1 was found to be overexpressed in tumor compared to normal tissue of CRC patients. In vitro, HKDC1-deficient human Caco-2 and murine CMT-93 cells displayed reduced proliferation, altered susceptibility to cell death induction, and disrupted mitochondrial functions, particularly mitochondrial respiration. These altered cancer hallmarks were then corroborated in HKDC1-deficient normal and tumor-derived ApcMin/+ intestinal organoids. Immunoprecipitation and mass spectometry proteomic analyses revealed interactions of HKDC1 with several mitochondria-related proteins. In vivo, two distinct mouse models demonstrated that epithelial deletion of HKDC1 protected from carcinogenesis. First, ApcMin/+-Hkdc1∆IEC mice showed mildly improved disease phenotypes in the colon accompanied with reduced numbers of Ki67-positive proliferating epithelial cells. Finally, HKDC1-deficient Caco-2 cells completely failed to form any tumor mass in a xenograft model when implanted into immunodeficient mice. Conclusions: We demonstrate that HKDC1 influences cancer cell proliferation and susceptibility to cell death, potentially through interactions with mitochondrial proteins that regulate membrane permeability, ultimately impacting intestinal carcinogenesis. Collectively, these findings highlight the significance of HKDC1 for CRC pathobiology, presenting it as a promising target for further investigation and potential therapeutic interventions. ### Competing Interest Statement PR reports stock ownership in Gerion Biotech GmbH and consulting fees from Takeda. All other authors declare no competing interests.

Sirtuins are deacetylases that are highly conserved throughout the animal kingdom. They act as metabolic sensors that coordinate cellular responses, allowing an adapted response to various stressors. Epithelial cells, especially those of the intestine, are directly exposed to a wide range of stressors. Together with the microbiota, they form a complex ecosystem with mutual influences. The significance of sirtuins in this complex system is still waiting to be clarified. Here, we show that a protein-restricted diet strongly increases the intestinal expression of sirtuin 4 (dSirt4), the only mitochondrial sirtuin in Drosophila. To elucidate the effects of deregulated dSirt4 expression in the intestine, we analyzed dSirt4 knockout flies. These flies showed substantial changes in their intestinal proteome and physiological properties. One of the most striking effects was the strong induction of lysozymes in the intestine, with a corresponding increase in lysozyme activity. This effect was organ-autonomous, as it was also observed in flies with dSirt4 knocked out only in intestinal enterocytes. The significant increase in lysozyme abundance in response to tissue-specific dSirt4 knockdown did not reduce the total number of bacteria in the intestine. However, it did affect the microbiota composition by reducing the number of gram-positive bacteria. This effect on microbiota composition can be attributed to dSirt4-dependent lysozyme expression, which is absent in a lysozyme-deficient background. dSirt4 knockout in the enterocytes shortened the lifespan of the flies, as did ectopic lysozyme overexpression in the enterocytes. The only mitochondrial sirtuin in Drosophila, dSirt4, is induced by dietary stress in intestinal epithelial cells, which directly regulates the lysozyme activity of these cells. We could associate this altered lysozyme activity with a shift in the microbiota composition, demonstrating a direct link between stress, nutrition, and the host’s microbiota regulation.

Phagocytosis is a conserved cellular mechanism for food uptake, defense, and animal-microbe interactions in metazoans. How the discrimination and subsequent processing of different microbes in marine invertebrates is facilitated remains largely unknown. Thereto, we combined a recently developed phagocytic assay with proteomics analysis to compare the phagocytic activity of the sponge Halichondria panicea upon encounter with the native Hal 281 (i.e., H. panicea isolate) and the foreign NJ 1 (i.e., Nematostella vectensis isolate) Vibrio. The sponge cell fraction was recovered after Vibrio exposure of 30 and 60 min and used for cellular (fluorescence-activated cell sorting and microscopy) and proteomics analyses. While the number of phagocytically active cells was similar between the isolates (P = 0.19), the distribution of vibrios over cell types differed (P = 0.02) over time, with the tendency for accumulation of NJ 1 in choanocyte-like cells compared to a shift of Hal 281 being incorporated from choanocyte-like to archaeocyte-like cells. Initially, both vibrios elicited a proteomic response related to bacterial infection and immunity (e.g., ADAM10, RAPTOR), followed by an increase of lysosomal and endocytic proteins (e.g., NPC2) after 60 min. The attenuation of the immune response and concomitant increase of vesicular trafficking in Hal 281 after 60 min corroborates cellular observations suggesting the fast transfer of Hal 281 from choanocyte-like cells to archaeocyte-like cells, compared to an accumulation of NJ 1 in the former. Subtle but distinct differences suggest strain-specific discrimination between the two tested vibrios and may indicate a degree of immune specificity in sponges.IMPORTANCEMetazoans recognize and discriminate between different microbes. In marine invertebrates, the underlying mechanisms of microbial discrimination and immune specificity are, however, not well understood. Phagocytosis is a conserved cellular process from amoeba to humans that facilitates the ingestion and digestion of microbial cells and likely plays a role in this discrimination. To elucidate the molecular and cellular basis of this microbial discrimination, we examined the differential phagocytic processing of a native (i.e., sponge-isolated) and foreign (i.e., sea anemone-isolate) Vibrio in a marine sponge. Our findings revealed that both vibrios provoke an initial bacterial infection- and immune-related, followed by a lysosomal- and endocytic-related proteomic response. Nuanced differences in the cellular and molecular processing suggest a strain-specific discrimination between the two vibrios. This study investigates a mechanism for microbial discrimination in an early-divergent metazoan and may provide a valuable model for studying the evolution of immunity and its role in animal-microbe interactions.

Until recently, small open reading frame (sORF)-encoded proteins of fewer than 100 amino acids, have attracted increasing attention over the past decade after being overlooked due to limitations in conventional detection methodologies. While numerous previously unannotated sORFs have recently been identified in the mesophilic archaeal model system Methanosarcina mazei , the physiological roles of most of their encoded small proteins remain unknown. We report here the functional characterization of sORF16 encoded small protein MtrR (49 amino acids) and show that it localizes oligomerically at the cytoplasmic membrane. There, it interacts with and influences the activity of tetrahydrosarcinapterin S-methyltransferase (Mtr), a key membrane-bound complex involved in energy metabolism. In vitro interaction and in vivo copurification assays revealed interactions between MtrR and the Mtr-complex, and microscale thermophoresis showed specific interactions with the MtrA subunit. Mutant strains lacking sORF16 exhibited significantly impaired growth in the presence of molecular hydrogen (H2), irrespective of the carbon source. We posit that by modulating the activity of the Mtr-complex, MtrR enables the archaeon adapt to changing environmental H2 conditions. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, RSCHM1052/20-1, RSCHM1052/20-2, TH872/10-1, TH872/10-2 Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, SCHU 3364/1-1 Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, RTG 2937 European Research Council, 101075992

Efficient separation is crucial for an in-depth analysis of proteoforms in complex samples. Two-dimensional (2D) separation platforms exhibit increased selectivity and peak capacity compared to their one-dimensional components. Here, we present for the first time a platform for online selective comprehensive coupling of reversed-phase nanoLC and capillary zone electrophoresis-mass spectrometry (CZE-MS) and its application for intact top-down (TD) proteoform analysis. The coupling of these techniques is promising since reversed-phase nanoLC allows for large-volume injection and separation of different proteins by hydrophobicity, whereas CZE-MS is powerful for the separation of proteoforms varying in charge and/or size. Combining reversed-phase nanoLC in the first dimension and highly efficient successive multiple ionic-polymer layers CZE-MS in the second dimension, we evaluated our setup by analyzing a human cell lysate. Using our 2D platform for intact TD proteoform characterization, we were able to detect two to four times more proteoforms compared to one-dimensional (1D) nanoLC-MS measurements in the sampled nanoLC time window. A targeted data evaluation approach for Histone H4 revealed additional proteoforms varying in acetylation and methylation, previously not detected using 1D nanoLC-MS. Combining the power of nanoLC and CZE, the here presented platform will advance the targeted proteoform analysis on the intact protein level in complex biological samples.

Innate immunity, traditionally viewed as non-specific, is increasingly recognized for its capacity to regulate microbial communities with precision. In the sea anemone Nematostella vectensis , we uncover a form of selective immunity mediated by nematosomes—motile immune cell clusters that preferentially phagocytose foreign Vibrio isolates while sparing native bacteria. We identify the transcription factor cJUN as essential for this process: CRISPR/Cas9-mediated knockout of cJUN impairs nematosome proliferation, reduces lysosomal activation, and alters microbiome composition by allowing colonization of non-native strains. These results link immune gene function to microbial selectivity and demonstrate that even early-diverging animals exhibit immune discrimination. Our findings challenge the classical dichotomy between innate and adaptive immunity and reveal that immune specificity may be evolutionarily ancient. This work establishes Nematostella as a model for studying microbiome-induced innate immune training and highlights conserved mechanisms that maintain host-microbe homeostasis.

The metalloprotease meprin β is upregulated in neurons and astrocytes of Alzheimer’s disease patients’ brains. While the role of meprin β as the β-secretase of amyloid precursor protein (APP) has been characterized, its broader substrate profile within the brain remains largely unexplored. Hence, to identify additional substrates, we conducted N-terminomics of brain lysates from mice overexpressing meprin β in astrocytes employing the Hydrophobic Tagging-Assisted N-terminal Enrichment (HYTANE) strategy. We observed 3906 (82.2%) N-terminal peptides and identified seven new substrates that match meprin β in terms of localization and cleavage specificity. Of note, the meprin β overexpressing mice show mild cognitive impairments caused by amyloidogenic APP processing alongside hyperactivity and altered exploratory behavior seemingly independent of APP cleavage. Hence, latrophilin-3 was of particular interest, as latrophilin-3 defects are associated with hyperactivity in mice and human. In brain lysates from mice overexpressing meprin β in astrocytes as well as in cellulo, we validated the cleavage of latrophilin-3, resulting in the release of two N-terminal domains. These domains promote interactions with neuronal proteins such as fibronectin leucine-rich repeat transmembrane proteins, promoting adequate synapse formation. Thus, meprin β might affect synaptic integrity by cleaving interaction domains of latrophilin-3, potentially exacerbating the observed hyperactivity phenotype.

Several steps of cancer progression, from tumor onset to metastasis, critically involve proteolytic activity. To elucidate the role of proteases in cancer, it is particularly important to consider single-nucleotide variants (SNVs) that affect the active site of proteases, thereby influencing cleavage specificity, substrate processing, and thus cancer cell behavior. To facilitate systematic studies, we here present a targeted approach to determine the impact of cancer-associated protease variants (TACAP). Starting with the semiautomated identification of potential specificity-modulating SNVs, our workflow comprises mass spectrometry–based cleavage specificity profiling and substrate identification, localization, and inhibitor studies, followed by functional analyses investigating cancer cell properties. To demonstrate the feasibility of TACAP, we analyzed the meprin β R238Q variant. This amino acid exchange R238Q leads to a loss of meprin β’s characteristic cleavage preference for acidic amino acids at P1′ position, accompanied with changes in substrate pool and inhibitor affinity compared to meprin β wild type.

Top-down analysis of intact proteins and middle-down analysis of proteins subjected to limited digestion require efficient detection of traces of proteoforms in samples, necessitating the reduction of sample complexity by thorough pre-fractionation of the proteome components in the sample. SDS–PAGE is a simple and inexpensive high-resolution protein-separation technique widely used in biochemical and molecular biology experiments. Although its effectiveness for sample preparation in bottom-up proteomics has been proven, establishing a method for highly efficient recovery of intact proteins from the gel matrix has long been a challenge for its implementation in top-down and middle-down proteomics. As a much-awaited solution to this problem, we present an experimental protocol for efficient proteoform fractionation from complex biological samples using passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry (PEPPI-MS), a rapid method for extraction of intact proteins separated by SDS–PAGE. PEPPI-MS allows recovery of proteins below 100 kDa separated by SDS–PAGE in solution with a median efficiency of 68 This protocol outlines a cost-effective and rapid protein sample pre-fractionation strategy based on highly efficient passive elution from gels, recovering intact proteins for top-down proteomics or partially digested proteins for middle-down proteomics.

Mass spectrometry-based top-down protein analysis requires efficient separation. In the context of proteoform analysis, capillary zone electrophoresis (CZE) is very valuable. The resolution of two peaks in CZE can be increased when the absolute mobility of the counter-directed electroosmotic flow (EOF) is close to the effective mobility of the analytes, resulting in a low apparent mobility of the analytes. The mobility of the EOF of highly efficient sulfobetaine-modified poly(α-L-lysine) (α-PLL) coatings changes depending on the number of modified side chains. Here, such coatings are used to selectively increase the peak resolution of proteoforms of model proteins and analytes in a complex protein sample (intact yeast protein extract). Whereas a high EOF system allows for the separation of proteins of a wide mobility range (complete proteome), lower EOF systems allow for a much better separation of proteins and proteoforms of low mobility, including those containing acidic post-translation modifications (PTMs). This leads to the identification of 2.5 times more proteoforms by MS/MS experiments in the lower mobility range of the yeast proteome. The sulfobetaine-modified α-PLL coatings presented here exhibit a toolbox for highly resolved separation of proteins and proteoforms in targeted or untargeted top-down protein analysis. SUMMARY: Sample complexity is one of the main challenges when analyzing a proteome on the proteoform level. In the course of this, capillary electrophoresis-mass spectrometry turned out to be an excellent tool because of its high-performing separation, particularly for large molecules. Here, we present a method enabling the best possible separation due to efficient and EOF-tunable coatings, allowing for flexible and dedicated selection of a range of proteins and proteoforms to be analyzed under ideal separation conditions. The high performance is demonstrated by the separation of proteoforms of common PTM-rich model proteins as well as complex proteome samples.

The following article for this Special Issue was published in an earlier Issue . A. Takemori, P. T. Kaulich, A. Tholey, and N. Takemori, “Dissolvable Polyacrylamide Gel Electrophoresis-Enabled High-Resolution Sample Fractionation for Middle-Down Proteomics” Proteomics 25 (2025): e70007. https://doi.org/10.1002/pmic.70007 . https://onlinelibrary.wiley.com/doi/10.1002/pmic.70007