Scarcity of high-quality organs, suboptimal organ quality assessment, unsatisfactory pre-implantation procedures, and poor long-term organ and patient survival are the main challenges currently faced by the solid organ transplant (SOT) field. New biomarkers for assessing graft quality pre-implantation, detecting, and predicting graft injury, rejection, dysfunction, and survival are critical to provide clinicians with invaluable prediction tools and guidance for personalized patients\' treatment. Additionally, new therapeutic targets are also needed to reduce injury and rejection and improve transplant outcomes. Proteins, which underlie phenotypes, are ideal candidate biomarkers of health and disease statuses and therapeutic targets. A protein can exist in different molecular forms, called proteoforms. As the function of a protein depends on its exact composition, proteoforms can offer a more accurate basis for connection to complex phenotypes than protein from which they derive. Mass spectrometry-based proteomics has been largely used in SOT research for identification of candidate biomarkers and therapeutic intervention targets by so-called \"bottom-up\" proteomics (BUP). However, such BUP approaches analyze small peptides in lieu of intact proteins and provide incomplete information on the exact molecular composition of the proteins of interest. In contrast, \"Top-down\" proteomics (TDP), which analyze intact proteins retaining proteoform-level information, have been only recently adopted in transplantation studies and already led to the identification of promising proteoforms as biomarkers for organ rejection and dysfunction. We anticipate that the use of top-down strategies in combination with new technological advancements in single-cell and spatial proteomics could drive future breakthroughs in biomarker and therapeutic target discovery in SOT.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

Snakebite envenomation has been a long-standing global issue that is difficult to treat, largely owing to the flawed nature of current immunoglobulin-based antivenom therapy and the complexity of snake venoms as sophisticated mixtures of bioactive proteins and peptides. Comprehensive characterisation of venom compositions is essential to better understanding snake venom toxicity and inform effective and rationally designed antivenoms. Additionally, a greater understanding of snake venom composition will likely unearth novel biologically active proteins and peptides that have promising therapeutic or biotechnological applications. While a bottom-up proteomic workflow has been the main approach for cataloguing snake venom compositions at the toxin family level, it is unable to capture snake venom heterogeneity in the form of protein isoforms and higher-order protein interactions that are important in driving venom toxicity but remain underexplored. This review aims to highlight the importance of understanding snake venom heterogeneity beyond the primary sequence, in the form of post-translational modifications that give rise to different proteoforms and the myriad of higher-order protein complexes in snake venoms. We focus on current top-down proteomic workflows to identify snake venom proteoforms and further discuss alternative or novel separation, instrumentation, and data processing strategies that may improve proteoform identification. The current higher-order structural characterisation techniques implemented for snake venom proteins are also discussed; we emphasise the need for complementary and higher resolution structural bioanalytical techniques such as mass spectrometry-based approaches, X-ray crystallography and cryogenic electron microscopy, to elucidate poorly characterised tertiary and quaternary protein structures. We envisage that the expansion of the snake venom characterisation \"toolbox\" with top-down proteomics and high-resolution protein structure determination techniques will be pivotal in advancing structural understanding of snake venoms towards the development of improved therapeutic and biotechnology applications.

One of the human proteome puzzles is an imbalance between the theoretically calculated and experimentally measured amounts of proteoforms. Considering the possibility of combinations of different post-translational modifications (PTMs), the quantity of possible proteoforms is huge. An estimation gives more than a million different proteoforms in each cell type. But, it seems that there is strict control over the production and maintenance of PTMs. Although the potential complexity of proteoforms due to PTMs is tremendous, available information indicates that only a small part of it is being implemented. As a result, a protein could have many proteoforms according to the number of modification sites, but because of different systems of personal regulation, the profile of PTMs for a given protein in each organism is slightly different.

Existing knowledge on changes of the haptoglobin (Hp) molecule suggests that it may exist in multiple proteoforms, which obviously exhibit different functions. Using two-dimensional electrophoresis (2DE) in combination with mass spectrometry and immunodetection, we have analyzed blood plasma samples from both healthy donors and patients with primary grade IV glioblastoma (GBM), and obtained a detailed composite 2DE distribution map of β-chain proteoforms, as well as the full-length form of Hp (zonulin). Although the total level of plasma Hp exceeded normal values in cancer patients (especially patients with GBM), the presence of particuar proteoforms, detected by their position on the 2DE map, was very individual. Variability was found in both zonulin and the Hp β-chain. The presence of an alkaline form of zonulin in plasma can be considered a conditional, but insufficient, GBM biomarker. In other words, we found that at the level of minor proteoforms of Hp, even in normal conditions, there was a high individual variability. On the one hand, this raises questions about the reasons for such variability, if it is present not only in Hp, but also in other proteins. On the other hand, this may explain the discrepancy between the number of experimentally detected proteoforms and the theoretically possible ones not only in Hp, but also in other proteins.

With growing recognition and acknowledgement of the genuine complexity of proteomes, we are finally entering the post-proteogenomic era. Routine assessment of proteomes as inferred correlates of gene sequences (i.e., canonical \'proteins\') cannot provide the necessary critical analysis of systems-level biology that is needed to understand underlying molecular mechanisms and pathways or identify the most selective biomarkers and therapeutic targets. These critical requirements demand the analysis of proteomes at the level of proteoforms/protein species, the actual active molecular players. Currently, only highly refined integrated or integrative top-down proteomics (iTDP) enables the analytical depth necessary to provide routine, comprehensive, and quantitative proteome assessments across the widest range of proteoforms inherent to native systems. Here we provide a broad perspective of the field, taking in historical and current realities, to establish a more balanced understanding of where the field has come from (in particular during the ten years since Proteomes was launched), current issues, and how things likely need to proceed if necessary deep proteome analyses are to succeed. We base this in our firm belief that the best proteomic analyses reflect, as closely as possible, the native sample at the moment of sampling. We also seek to emphasise that this and future analytical approaches are likely best based on the broad recognition and exploitation of the complementarity of currently successful approaches. This also emphasises the need to continuously evaluate and further optimize established approaches, to avoid complacency in thinking and expectations but also to promote the critical and careful development and introduction of new approaches, most notably those that address proteoforms. Above all, we wish to emphasise that a rigorous focus on analytical quality must override current thinking that largely values analytical speed; the latter would certainly be nice, if only proteoforms could thus be effectively, routinely, and quantitatively assessed. Alas, proteomes are composed of proteoforms, not molecular species that can be amplified or that directly mirror genes (i.e., \'canonical\'). The problem is hard, and we must accept and address it as such, but the payoff in playing this longer game of rigorous deep proteome analyses is the promise of far more selective biomarkers, drug targets, and truly personalised or even individualised medicine.

Zebra mussel (ZM), Dreissena polymorpha, commonly used as a sentinel species in freshwater biomonitoring, is now in competition for habitat with quagga mussel (QM), Dreissena rostriformis bugensis. This raises the question of the quagga mussel\'s use in environmental survey. To better characterise QM response to stress compared with ZM, both species were exposed to cadmium (100 µg·L-1), a classic pollutant, for 7 days under controlled conditions. The gill proteomes were analysed using two-dimensional electrophoresis coupled with mass spectrometry. For ZM, 81 out of 88 proteoforms of variable abundance were identified using mass spectrometry, and for QM, 105 out of 134. Interestingly, the proteomic response amplitude varied drastically, with 5.6% of proteoforms of variable abundance (DAPs) in ZM versus 9.4% in QM. QM also exhibited greater cadmium accumulation. Only 12 common DAPs were observed. Several short proteoforms were detected, suggesting proteolysis. Functional analysis is consistent with the pleiotropic effects of the toxic metal ion cadmium, with alterations in sulphur and glutathione metabolisms, cellular calcium signalling, cytoskeletal dynamics, energy production, chaperone activation, and membrane events with numerous proteins involved in trafficking and endocytosis/exocytosis processes. Beyond common responses, the sister species display distinct reactions, with cellular response to stress being the main category involved in ZM as opposed to calcium and cytoskeleton alterations in QM. Moreover, QM exhibited greater evidence of proteolysis and cell death. Overall, these results suggest that QM has a weaker stress response capacity than ZM.

Polypeptides generated from proteolytic processing of protein precursors, or proteolytic proteoforms, play an important role in diverse biological functions and diseases. However, their often-small size and intricate post-translational biogenesis preclude the use of simple genetic tagging in their cellular studies. Herein, we develop a labeling strategy for this class of proteoforms, based on residue-specific genetic code expansion labeling with a molecular beacon design. We demonstrate the utility of such a design by creating a molecular beacon reporter to detect amyloid-β peptides, known to be involved in the pathogenesis of Alzheimer\'s disease, as they are produced from amyloid precursor protein (APP) along the endocytic pathway of living cells.
Biogenesis of amyloid‐β peptides from their precursor protein can be tracked in endocytic compartments of living cells by a molecular beacon labeling strategy based on genetic code expansion.

Monoclonal antibodies (mAbs) are an essential class of biotherapeutics. A platform process is used for mAb development to ensure clinically safe and stable molecules. Regulatory authorities ensure that mAb production processes include sufficient viral clearance steps to achieve less than one virus particle per million doses of product. Virus filtration is used for size-based removal of enveloped and nonenveloped viruses during downstream processing of mAbs. Process development in mAb purification relies on empirical approaches and often includes adsorptive prefiltration to mitigate virus filter fouling. Opportunities for molecular-level prediction of mAb filterability are needed to plug the existing knowledge gap in downstream processing. A molecular-level approach to understanding the factors influencing mAb filterability may reduce process development time, material loss, and processing costs due to oversized virus filters. In this work, pH step gradient fractionation was applied on polished bulk mAb feed to obtain concentrated pools of fractionated mAb variants. Biophysical properties and quality attributes of fractionated pools, including oligomeric state (size), isoelectric point profile, diffusion interaction parameters, and glycoform profile, were determined using bioanalytical methods. Filterability (loading and throughput) of fractionated pools were evaluated. Statistical methods were used to obtain correlations between quality attributes of mAb fractions and filterability on the Viresolve Pro virus filter.

The misfolding and aggregation of α-synuclein is linked to a family of neurodegenerative disorders known as synucleinopathies, the most prominent of which is Parkinson\'s disease (PD). Understanding the aggregation process of α-synuclein from a mechanistic point of view is thus of key importance. SNCA, the gene encoding α-synuclein, comprises six exons and produces various isoforms through alternative splicing. The most abundant isoform is expressed as a 140-amino acid protein (αSyn-140), while three other isoforms, αSyn-126, αSyn-112, and αSyn-98, are generated by skipping exon 3, exon 5, or both exons, respectively. In this study, we performed a detailed biophysical characterization of the aggregation of these four isoforms. We found that αSyn-112 and αSyn-98 exhibit accelerated aggregation kinetics compared to αSyn-140 and form distinct aggregate morphologies, as observed by transmission electron microscopy. Moreover, we observed that the presence of relatively small amounts of αSyn-112 accelerates the aggregation of αSyn-140, significantly reducing the aggregation half-time. These results indicate a potential role of alternative splicing in the pathological aggregation of α-synuclein and provide insights into how this process could be associated with the development of synucleinopathies.