Bridging the Gap From Proteomics Technology to Clinical Application: Highlights From the 68th Benzon Foundation Symposium.

The 68th Benzon Foundation Symposium brought together leading experts to explore the integration of mass spectrometry-based proteomics and artificial intelligence to revolutionize personalized medicine. This report highlights key discussions on recent technological advances in mass spectrometry-based proteomics, including improvements in sensitivity, throughput, and data analysis. Particular emphasis was placed on plasma proteomics and its potential for biomarker discovery across various diseases.

The Circulating Proteome─Technological Developments, Current Challenges, and Future Trends.

Recent improvements in proteomics technologies have fundamentally altered our capacities to characterize human biology. There is an ever-growing interest in using these novel methods for studying the circulating proteome, as blood offers an accessible window into human health. However, every methodological innovation and analytical progress calls for reassessing our existing approaches and routines to ensure that the new data will add value to the greater biomedical research community and avoid previous errors.

Consistency across multi-omics layers in a drug-perturbed gut microbial community.

Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics).

Profiling the human intestinal environment under physiological conditions.

The spatiotemporal structure of the human microbiome, proteome and metabolome reflects and determines regional intestinal physiology and may have implications for disease. Yet, little is known about the distribution of microorganisms, their environment and their biochemical activity in the gut because of reliance on stool samples and limited access to only some regions of the gut using endoscopy in fasting or sedated individuals. To address these deficiencies, we developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion.

Immobility-associated thromboprotection is conserved across mammalian species from bear to human.

Venous thromboembolism (VTE) comprising deep venous thrombosis and pulmonary embolism is a major cause of morbidity and mortality. Short-term immobility-related conditions are a major risk factor for the development of VTE. Paradoxically, long-term immobilized free-ranging hibernating brown bears and paralyzed spinal cord injury (SCI) patients are protected from VTE.

Transparent Exploration of Machine Learning for Biomarker Discovery from Proteomics and Omics Data.

Biomarkers are of central importance for assessing the health state and to guide medical interventions and their efficacy; still, they are lacking for most diseases. Mass spectrometry (MS)-based proteomics is a powerful technology for biomarker discovery but requires sophisticated bioinformatics to identify robust patterns. Machine learning (ML) has become a promising tool for this purpose.

Noninvasive proteomic biomarkers for alcohol-related liver disease.

Alcohol-related liver disease (ALD) is a major cause of liver-related death worldwide, yet understanding of the three key pathological features of the disease-fibrosis, inflammation and steatosis-remains incomplete. Here, we present a paired liver-plasma proteomics approach to infer molecular pathophysiology and to explore the diagnostic and prognostic capability of plasma proteomics in 596 individuals (137 controls and 459 individuals with ALD), 360 of whom had biopsy-based histological assessment. We analyzed all plasma samples and 79 liver biopsies using a mass spectrometry (MS)-based proteomics workflow with short gradient times and an enhanced, data-independent acquisition scheme in only 3 weeks of measurement time.

Dynamic human liver proteome atlas reveals functional insights into disease pathways.

Deeper understanding of liver pathophysiology would benefit from a comprehensive quantitative proteome resource at cell type resolution to predict outcome and design therapy. Here, we quantify more than 150,000 sequence-unique peptides aggregated into 10,000 proteins across total liver, the major liver cell types, time course of primary cell cultures, and liver disease states. Bioinformatic analysis reveals that half of hepatocyte protein mass is comprised of enzymes and 23% of mitochondrial proteins, twice the proportion of other liver cell types.

A knowledge graph to interpret clinical proteomics data.

Implementing precision medicine hinges on the integration of omics data, such as proteomics, into the clinical decision-making process, but the quantity and diversity of biomedical data, and the spread of clinically relevant knowledge across multiple biomedical databases and publications, pose a challenge to data integration. Here we present the Clinical Knowledge Graph (CKG), an open-source platform currently comprising close to 20 million nodes and 220 million relationships that represent relevant experimental data, public databases and literature. The graph structure provides a flexible data model that is easily extendable to new nodes and relationships as new databases become available.

Advances and Utility of the Human Plasma Proteome.

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods.

High-resolution serum proteome trajectories in COVID-19 reveal patient-specific seroconversion.

A deeper understanding of COVID-19 on human molecular pathophysiology is urgently needed as a foundation for the discovery of new biomarkers and therapeutic targets. Here we applied mass spectrometry (MS)-based proteomics to measure serum proteomes of COVID-19 patients and symptomatic, but PCR-negative controls, in a time-resolved manner. In 262 controls and 458 longitudinal samples of 31 patients, hospitalized for COVID-19, a remarkable 26% of proteins changed significantly.

Cohort profile: the MUNICH Preterm and Term Clinical study (MUNICH-PreTCl), a neonatal birth cohort with focus on prenatal and postnatal determinants of infant and childhood morbidity.

Purpose: The MUNICH Preterm and Term Clinical (MUNICH-PreTCl) birth cohort was established to uncover pathological processes contributing to infant/childhood morbidity and mortality. We collected comprehensive medical information of healthy and sick newborns and their families, together with infant blood samples for proteomic analysis. MUNICH-PreTCl aims to identify mechanism-based biomarkers in infant health and disease to deliver more precise diagnostic and predictive information for disease prevention.

A New Parallel High-Pressure Packing System Enables Rapid Multiplexed Production of Capillary Columns.

Reversed-phase HPLC is the most commonly applied peptide-separation technique in MS-based proteomics. Particle-packed capillary columns are predominantly used in nanoflow HPLC systems. Despite being the broadly applied standard for many years, capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems.

Molecular Origin of Blood-Based Infrared Spectroscopic Fingerprints*.

Infrared spectroscopy of liquid biopsies is a time- and cost-effective approach that may advance biomedical diagnostics. However, the molecular nature of disease-related changes of infrared molecular fingerprints (IMFs) remains poorly understood, impeding the method's applicability. Here we probe 148 human blood sera and reveal the origin of the variations in their IMFs.

Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers.

The correspondence of cell state changes in diseased organs to peripheral protein signatures is currently unknown. Here, we generated and integrated single-cell transcriptomic and proteomic data from multiple large pulmonary fibrosis patient cohorts. Integration of 233,638 single-cell transcriptomes (n = 61) across three independent cohorts enabled us to derive shifts in cell type proportions and a robust core set of genes altered in lung fibrosis for 45 cell types.

Ethical Principles, Constraints and Opportunities in Clinical Proteomics.

Recent advances in mass spectrometry (MS)-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with impacts on the health, privacy, and wellbeing of individuals.

Plasma Proteomes Can Be Reidentifiable and Potentially Contain Personally Sensitive and Incidental Findings.

The goal of clinical proteomics is to identify, quantify, and characterize proteins in body fluids or tissue to assist diagnosis, prognosis, and treatment of patients. In this way, it is similar to more mature omics technologies, such as genomics, that are increasingly applied in biomedicine. We argue that, similar to those fields, proteomics also faces ethical issues related to the kinds of information that is inherently obtained through sample measurement, although their acquisition was not the primary purpose.

Ethical principles, opportunities and constraints in clinical proteomics.

Recent advances in MS-based proteomics have vastly increased the quality and scope of biological information that can be derived from human samples. These advances have rendered current workflows increasingly applicable in biomedical and clinical contexts. As proteomics is poised to take an important role in the clinic, associated ethical responsibilities increase in tandem with the impact on the health, privacy, and well-being of individuals.

The proteome landscape of the kingdoms of life.

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported, advances in mass-spectrometry-based proteomics have enabled increasingly comprehensive identification and quantification of the human proteome. However, there have been few comparisons across species, in stark contrast with genomics initiatives.

Proteome profiling in cerebrospinal fluid reveals novel biomarkers of Alzheimer's disease.

Neurodegenerative diseases are a growing burden, and there is an urgent need for better biomarkers for diagnosis, prognosis, and treatment efficacy. Structural and functional brain alterations are reflected in the protein composition of cerebrospinal fluid (CSF). Alzheimer's disease (AD) patients have higher CSF levels of tau, but we lack knowledge of systems-wide changes of CSF protein levels that accompany AD.

Multiparametric Assays for Accelerating Early Drug Discovery.

Drug discovery campaigns are hampered by substantial attrition rates largely due to a lack of efficacy and safety reasons associated with candidate drugs. This is true in particular for genetically complex diseases, where insufficient knowledge of the modulatory actions of candidate drugs on targets and entire target pathways further adds to the problem of attrition. To better profile compound actions on targets, potential off-targets, and disease-linked pathways, new innovative technologies need to be developed that can elucidate the complex cellular signaling networks in health and disease.

Proteomics of Cytochrome c Oxidase-Negative versus -Positive Muscle Fiber Sections in Mitochondrial Myopathy.

The mosaic distribution of cytochrome c oxidase (COX) and COX muscle fibers in mitochondrial disorders allows the sampling of fibers with compensated and decompensated mitochondrial function from the same individual. We apply laser capture microdissection to excise individual COX and COX fibers from the biopsies of mitochondrial myopathy patients. Using mass spectrometry-based proteomics, we quantify >4,000 proteins per patient.

Mass Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data.

The proteomic analysis of human blood and blood-derived products (e.g., plasma) offers an attractive avenue to translate research progress from the laboratory into the clinic.

Plasma Proteome Profiling to detect and avoid sample-related biases in biomarker studies.

Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)-based proteomics now allows highly specific and quantitative readout of the plasma proteome.

Plasma proteome profiling discovers novel proteins associated with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) affects 25% of the population and can progress to cirrhosis with limited treatment options. As the liver secretes most of the blood plasma proteins, liver disease may affect the plasma proteome. Plasma proteome profiling of 48 patients with and without cirrhosis or NAFLD revealed six statistically significantly changing proteins (ALDOB, APOM, LGALS3BP, PIGR, VTN, and AFM), two of which are already linked to liver disease.