Implementing proteomics-based solutions has emerged as a potentially highly powerful and transformative approach in enhancing patient management by streamlining clinical decision-making and accelerating drug development. Specific changes in proteins are generally responsible for onset and progression of disease. This understanding serves as a foundation for identifying biomarkers, discovering drug targets, and developing novel therapeutics. Despite substantial progress, several challenges and gaps persist. These include among others technical limitations, regulatory hurdles, legal, ethics and data protection issues. Addressing these obstacles necessitates a multifaceted approach involving critical evaluation, cross-disciplinary collaboration, and the inclusion of diverse stakeholder perspectives. Such a comprehensive strategy appears essential for advancing proteomics from research to implementation, ultimately benefiting patients. To this end, the PROTEOMICS journal is launching a special section dedicated to clinical proteomics. This initiative aims to highlight the critical aspects of (prote)omics-guided biomarker and drug development, address existing challenges, and propose potential solutions for implementation. By fostering collaboration among scientists, clinicians, patients, regulators, insurance companies, pharmaceutical companies, and funding agencies, we hope to spark discussions that pave the way for adopting proteomics-based approaches to improve patient outcomes.

The clinical proteomics field has seen enormous growth in the past 20 years, with over 40000 scientific manuscripts published. At the same time, actual clinical application of the reported findings is obviously scarce. In this manuscript we discuss the key issues that may be responsible for this apparent lack of success, which cannot be measured by the numbers of publications, but by the impact on patient management and treatment. We proceed with suggestions for potential solutions, which include keeping a strict focus on potential patient benefit. We hope this manuscript can help shaping the field, so it can in fact deliver on its realistic promise, to bring significant improvement in management and care to patients.

Fluctuating salinity is symptomatic of climate change challenging aquatic species. The melting of polar ice, rising sea levels, coastal surface and groundwater salinization, and increased evaporation in arid habitats alter salinity world-wide. Moreover, the frequency and intensity of extreme weather events such as rainstorms and floods increase, causing rapid shifts in brackish and coastal habitat salinity. Such salinity alterations disrupt homeostasis, and ultimately diminish fitness, of aquatic organisms by interfering with metabolism, reproduction, immunity, and other critical aspects of physiology. Proteins are central for these physiological mechanisms. They represent the molecular building blocks of phenotypes that govern organismal responses to environmental challenges. Environmental cues regulate proteins in concerted fashion, necessitating holistic analyses of proteomes for comprehending salinity stress responses. Proteomics approaches reveal molecular causes of population declines and enable holistic bioindication geared towards timely interventions to prevent local extinctions. Proteomics analyses of salinity effects on aquatic organisms have been performed since the mid-1990s, propelled by the invention of two-dimensional protein gels, soft ionization techniques for mass spectrometry, and nano-liquid chromatography in the 1970s and 1980s. This review summarizes the current knowledge on salinity regulation of proteomes from aquatic organisms, including key methodological advances over the past decades.

Marine plastispheres represent dynamic microhabitats where microorganisms colonise plastic debris and interact. Metaproteomics has provided novel insights into the metabolic processes within these communities, however the early metabolic interactions driving the plastisphere formation remain unclear. This study utilised metaproteomic and metagenomic approaches to explore early plastisphere formation on low-density polyethylene (LDPE) over three (D3) and seven (D7) days, focusing on microbial diversity, metabolic activity, and biofilm development. In total, 2,948 proteins were analysed, revealing dominant proteomes from Pseudomonas and Marinomonas, with near-complete metagenome-assembled genomes. Pseudomonas dominated at D3, while at D7, Marinomonas, along with Acinetobacter, Vibrio, and other genera became more prevalent. Pseudomonas and Marinomonas showed high expression of reactive oxygen species (ROS) suppression proteins, associated with oxidative stress regulation, while granule formation, and alternative carbon utilisation enzymes, also indicated nutrient limitations. Interestingly, 13 alkane and other xenobiotic degradation enzymes were expressed by five genera. The expression of toxins, several type VI secretion system (TVISS) proteins, and biofilm formation proteins by Pseudomonas indicated their competitive advantage against other taxa. Upregulated metabolic pathways, including those relating to substrate transport also suggested enhanced nutrient cross-feeding within the biofilm. These insights enhance our understanding of plastisphere ecology and its potential for biotechnological applications.

Fertilisation is based on the tight interaction between sperm and egg and both sperm and egg proteomes are known to evolve and diverge rapidly even between closely related species. Understanding the sperm proteome therefore provides key insights into the mechanisms involved during fertilisation and the fusion between sperm and egg. Despite being a commonly used model organism for reproductive research, very little is currently understood about the zebrafish Danio rerio sperm proteome. We performed in depth proteomics analysis with six pooled samples containing ejaculates from ten males each. We confidently identified 5410 proteins, from which a total of 3900 GeneIDs were generated leading to 1720 Gene Ontology terms. This number of proteins is larger than the number of proteins described in sperm proteomes of other species.

Teleost fishes are a highly diverse and ecologically essential group of aquatic vertebrates and include coho salmon, Onchorhynchus kisutch. Coho are semelparous and all ovarian follicles develop synchronously. Owing to their ubiquitous distribution, teleost provide critical sources of food worldwide through subsistence, commercial fisheries, and aquaculture. Enhancement of commercial hatchery practices requires a detailed knowledge of teleost reproductive physiology. Despite decades of research on teleost reproductive processes, an in-depth proteome of teleost ovarian development has yet to be generated. We describe a coho salmon ovarian proteome of over 5700 proteins, generated with data independent acquisition, revealing the suite of detectable proteins that change through the transition from primary to secondary ovarian follicle development. This transition is critical for puberty onset, egg quality, and further embryonic development. Primary ovarian follicle development was marked by differential abundances of proteins involved in carbohydrate metabolism, protein turnover, and the complement pathway, suggesting elevated metabolism as the oocytes enter maturation. The greatest proteomic shift occurred during the transition from primary to secondary follicle growth, with increased abundance of proteins underlying cortical alveoli formation, extracellular matrix reorganization, iron binding, and cell-cell signaling. This work provides a foundation for identifying biomarkers of salmon oocyte stage and quality.

With an aging population, there has been significant progress in the discovery and measurement of bone turnover biomarkers since the 2000s, especially for monitoring skeletal diseases like osteoporosis. Multiple markers derived from type I collagen, such as CTX, NTX, PINP, and ICTP, have been developed. Extensive efforts have been devoted to characterizing these molecules; however, their complex crosslinked structures have posed significant analytical challenges, and to date, these biomarkers remain poorly characterized. Previous attempts at characterization involved gel-based separation methods and MALDI-TOF analysis on collagen peptides directly extracted from bone. However, using bone powder, while rich in collagen, does not represent the true structure of the peptides in the biofluids. In this study, our goal was to characterize plasma and serum CTX for subsequent LC-MS/MS method development. We extracted and characterized type I collagen peptides directly from human plasma and serum using a proteomics workflow that integrates preparative liquid chromatography, affinity chromatography, and high-resolution mass spectrometry. Subsequently, we successfully identified numerous CTX species, providing valuable insights into the characterization of these crucial biomarkers.

Single-cell proteomics (SCP) has advanced significantly in recent years, with new tools that have been specifically designed for preparation and analysis of single cells now commercially available to researchers. The field is sufficiently mature to be broadly accessible to any lab capable of isolating single cells and performing bulk-scale proteomic analyses. In this review, we highlight recent work in the SCP field that has significantly lowered the barrier to entry, thus providing a practical guide for those who are newly entering the SCP field. We outline the fundamental principles and report multiple paths to accomplish the key steps of a successful SCP experiment including sample preparation, separation and mass spectrometry data acquisition and analysis. We recommend that researchers start with a label-free SCP workflow, as achieving high-quality and quantitatively accurate results is more straightforward than label-based multiplexed strategies. By leveraging these accessible means, researchers can confidently perform SCP experiments and make meaningful discoveries at the single-cell level.

The human gut microbiota (GM) is a community of microorganisms that reside in the gastrointestinal tract. Recognized as a critical element of human health, the functions of the GM extend beyond gastrointestinal well-being to influence overall systemic health and susceptibility to disease. Among the other omic sciences, metaproteomics highlights additional facets that make it a highly valuable discipline in the study of GM. Indeed, it allows the protein inventory of complex microbial communities; peptides identified and quantified by liquid chromatography coupled to mass spectrometry analyses and then are mapped to the most likely proteins, taxa and functions by querying specific databases. The aim of this review was to compile comprehensive information on metaproteomic studies of the human GM, with a focus on the bacterial component, to assist newcomers in understanding the methods and types of research conducted in this field. The review outlines key steps in a metaproteomic-based study, such as protein extraction, database selection, and bioinformatic workflow. The importance of standardization is emphasized. In addition, a list of previously published studies is provided as hints for researchers interested in investigating the role of GM in health and disease states.

Human males and females show differences in the incidence of neutrophil-associated diseases and differences in neutrophil responses such as a faster response to the chemorepellent SLIGKV in males. Little is known about the basis of sex-based differences in human neutrophils. We used mass spectrometry to identify proteins and phosphoproteins in unstimulated human neutrophils and in neutrophils incubated with the SLIGKV, a protease activated receptor 2 agonist. There were 132 proteins with higher levels in unstimulated male neutrophils; these proteins tended to be associated with RNA regulation, ribosome, and phosphoinositide signaling pathways, whereas 30 proteins with higher levels in unstimulated female neutrophils were associated with metabolic processes, proteosomes, and phosphatase regulatory proteins. Unstimulated male neutrophils had increased phosphorylation of 32 proteins compared to females. After exposure to SLIGKV, male neutrophils showed a faster response in terms of protein phosphorylation compared to female neutrophils. Male neutrophils have higher levels of proteins and higher phosphorylation of proteins associated with RNA processing and signaling pathways. Female neutrophils have higher levels of proteins associated with metabolism and proteolytic pathways. This suggests that male neutrophils might be more ready to adapt to a new environment, and female neutrophils might be more effective at responding to pathogens.

LC-MS intact mass analysis and LC-MS/MS peptide mapping are foundational assays for developing biologic drugs and other commercial protein products. Certain PTM types, such as truncation and oxidation, increase the difficulty of precise proteoform characterization owing to inherent limitations in peptide and intact protein analyses. Top-down MS (TDMS) can resolve this ambiguity via fragmentation of specific proteoforms. We optimized our existing flow-programmed denaturing online buffer exchange ((fp)dOBE) approach to improve ESI sensitivity and increase TDMS sampling time for industrial applications. Using bovine alpha-lactalbumin (αLac), we tested data-dependent (DDA) and targeted strategies with 14 different MS/MS scan types featuring combinations of collisional- and electron-based fragmentation as well as proton transfer charge reduction. This large dataset was processed using a new software platform, named TDAcquireX, that improves proteoform characterization through TDMS data aggregation. (fp)dOBE-based DDA-TDMS analysis readily identified truncated proteoforms. For targeted TDMS, we used Sliding Window fragment ion deconvolution to analyze composite proteoform (cPrSM) results. This strategy facilitates probability-based noise filtering of individual fragments, simultaneously increasing matched fragments while decreasing total fragment masses. Using this strategy, we characterized oxidation positional isomers on αLac, finding ETD fragmentation uniquely provided accurate relative occupancy ratios by oxidation-specific challenges.

Single-cell proteomics (SCP) aims to characterize the proteome of individual cells, providing insights into complex biological systems. It reveals subtle differences in distinct cellular populations that bulk proteome analysis might overlook, which is essential for understanding disease mechanisms and developing targeted therapies. Mass spectrometry (MS) methods in SCP allow the identification and quantification of thousands of proteins from individual cells and this review highlights the role of data-independent acquisition MS (DIA-MS) in SCP. One major hurdle in SCP is the limited material in single-cell samples, but DIA-based techniques offer multiple potential solutions for their analysis. Utilizing wide precursor isolation windows to fragment multiple peptides simultaneously, DIA-based methods improve sensitivity, quantitative accuracy, and reproducibility at a cost in data analysis complexity. DIA methods can also be combined with sample multiplexing methods to increase the sample throughput, currently a key limitation in SCP. Challenges remain for interpreting sample multiplexed data from DIA-based SCP experiments, particularly with regards to isobaric tagging methods. Even still, we believe that DIA-based SCP approaches will play a major role in our understanding of systems biology.

Targeted proteomics, which includes parallel reaction monitoring (PRM), is typically utilized for more precise detection and quantitation of key proteins and/or pathways derived from complex discovery proteomics datasets. Initial discovery-based analysis using data independent acquisition (DIA) can obtain deep proteome coverage with low data missingness while targeted PRM assays can provide additional benefits in further eliminating missing data and optimizing measurement precision. However, PRM method development from bioinformatic predictions can be tedious and time-consuming because of the DIA output complexity. We address this limitation with a Python script that rapidly generates a PRM method for the TIMS-TOF platform using DIA data and a user-defined target list. To evaluate the script, DIA data generated from HeLa cell lysate (200 ng, 45-minute gradient method) as well as canonical pathway information from Ingenuity Pathway Analysis was utilized to generated a pathway-driven PRM method. Subsequent PRM analysis of targets within the example pathway, regulation of apoptosis, resulted in improved chromatographic data and enhanced quantitation precision (100% peptides below 10% CV with a median CV of 2.9%, n=3 technical replicates). The script is freely available at https://github.com/StevensOmicsLab/PRM-script and provides a framework that can be adapted to multiple DDA/DIA data outputs and instrument-specific PRM method types.

Sequencing the tyrosine phosphoproteome using MS-based proteomics is challenging due to the low abundance of tyrosine phosphorylation in cells, a challenge compounded in scarce samples like primary cells or clinical samples. The broad-spectrum optimization of selective triggering (BOOST) method was recently developed to increase phosphotyrosine sequencing in low protein input samples by leveraging tandem mass tags (TMT), phosphotyrosine enrichment, and a phosphotyrosine-loaded carrier channel. Here, we demonstrate the viability of BOOST in T cell receptor (TCR)-stimulated primary murine T cells by benchmarking the accuracy and precision of the BOOST method and discerning significant alterations in the phosphoproteome associated with receptor stimulation. Using 1 milligram of protein input (about 20 million cells) and BOOST, we identify and precisely quantify more than 2,000 unique pY sites compared to about 300 unique pY sites in non-BOOST control samples. We show that although replicate variation increases when using the BOOST method, BOOST does not jeopardise quantitative precision or the ability to determine statistical significance for peptides measured in triplicate. Many pY previously uncharacterised sites on important T cell signalling proteins are quantified using BOOST, and we identify new TCR responsive pY sites observable only with BOOST. Finally, we determine that the phase-spectrum deconvolution method on Orbitrap instruments can impair pY quantitation in BOOST experiments.

Alcohol consumption and high-fat diets often coincide in Western society, exerting negative synergistic effects on the liver. While many studies have demonstrated the impact of ALD and NAFLD on organ protein expression, none have offered a comprehensive view of the dysregulation at the level of the membrane proteome. In this study, we utilize peptidisc and solvent precipitation (SP4) methods to isolate and compare the membrane protein content of the liver with its unique biological functions. Using mice treated with a high-fat diet and ethanol in drinking water, we identified 1,563 liver proteins, with 46% predicted to have a transmembrane segment. Among these, 106 integral membrane proteins are dysregulated compared to the untreated sample. Gene ontology analysis reveals several dysregulated membrane processes associated with lipid metabolism, cell adhesion, xenobiotic processing, and mitochondrial membrane formation. Pathways related to cholesterol and bile acid transport are also mutually affected, suggesting an adaptive mechanism to counter the steatosis of the liver model. Our peptidisc-based membrane proteome profiling thus emerges as an effective way to gain insights into the role of the transmembrane proteome in disease development, warranting further in-depth analysis of the individual effect of the identified dysregulated membrane proteins.

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signaling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g. HB-EGF) are known to bind to heparan sulfate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e. HB-EGF, CX3CL1) and 17 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4,930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3,426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were unique in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate’s deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Predicting protein function from protein sequence, structure, interaction, and other relevant information is important for generating hypotheses for biological experiments and studying biological systems, and therefore has been a major challenge in protein bioinformatics. Numerous computational methods had been developed to advance protein function prediction gradually in the last two decades. Particularly, in the recent years, leveraging the revolutionary advances in artificial intelligence (AI), more and more deep learning methods have been developed to improve protein function prediction at a faster pace. Here, we provide an in-depth review of the recent developments of deep learning methods for protein function prediction. We summarize the significant advances in the field, identify several remaining major challenges to be tackled, and suggest some potential directions to explore. The data sources and evaluation metrics widely used in protein function prediction are also discussed to assist the machine learning, AI, and bioinformatics communities to develop more cutting-edge methods to advance protein function prediction.