Evolution of Mass Spectrometry Instruments and Techniques for Blood Proteomics.

Report on the Barbados Workshop

Consumer concerns regarding food nutrition and quality are becoming increasingly prevalent. High-resolution mass spectrometry (HRMS)-based metabolomics stands as a cutting-edge and widely embraced technique in the realm of food component analysis and detection. It boasts the capability to identify character metabolites at exceedingly low abundances, which remain undetectable by conventional platforms. It can also enable real-time monitoring of the flux of targeted compounds in metabolic synthesis and decomposition. With the emergence of artificial intelligence and machine learning, it has become more convenient to process the vast data sets of metabolomics and identify biomarkers. The review summarizes the latest applications of HRMS-based metabolomics platforms in traditional foods, novel foods, and pharmaceutical-food homologous matrices. It compares the suitability of HRMS to nuclear magnetic resonance (NMR) in metabolomics across three dimensions and discusses the principles and application scenarios of various mass spectrometry technologies.

Medical Applications of Mass Spectrometry

Substantial efforts are currently invested in development of micro- and nanofluidics-based systems as front end technology for electrospray ionization (ESI) mass spectrometry (MS). Since its first introduction in biological MS chip-based ESI demonstrated a high potential to discover novel biopolymer species due to the efficient ionization properties, preferential formation of multiply charged ions and the elevated reproducibility and sensitivity. In combination with high-resolution mass spectrometers or instruments able to perform multistage fragmentation, chip-electrospray confirmed its unique ability to offer structural elucidation of minor species in complex mixtures, which often represent valuable biomarkers of severe diseases. This aspect is of particular importance for the applicability of chip MS in clinical investigation where only minute amounts of biological material are available. In view of these major advantages of nanofluidics in conjunction with modern MS, this chapter reviews the strategies, which allowed a successful application of chip technology for early diagnostic of lysosomal storage diseases. The first part is dedicated to the principles of ESI MS and to advanced nanochip systems for ESI MS. The second part highlights important achievements of nanochip ESI MS in biomarker discovery and diagnostic of LSDs such as Fabry and Schindler diseases. Finally, this chapter emphasizes that advanced chip ESI MS has real perspectives to become a routine method for early diagnosis and therapy of severe pathologies such as LSDs.

Immunoproteomics is the branch of proteomics with an emphasis on the study of functional peptides and proteins related to the immune system. Combining proteomics techniques with immunological research aims to uncover the complex interactions of proteins involved in immune responses. This review discusses the methods, applications, and recent advancements in immunoproteomics, highlighting its critical role in understanding immune responses, discovering biomarkers, and developing vaccines and therapeutics. This study offers a comprehensive exploration of the methodologies, applications, and advancements within immunoproteomics. Techniques such as mass spectrometry, antibody-based assays, and computational analysis are pivotal in unraveling the complexities of the immune system at the protein level. Immunoproteomics finds diverse applications in biomarker discovery, vaccine development, autoimmune disease research, infectious disease diagnostics, and cancer immunotherapy. Challenges, including data integration, sample heterogeneity, and biomarker validation, persist, necessitating innovative approaches and interdisciplinary collaborations. In the future, immunoproteomics will likely play a major role in expanding our knowledge of immune-related diseases and accelerating the creation of targeted and precise immunotherapies.

Clinical proteomics involves the analysis of protein expression of disease proteomes, with the aim of solving a specific clinical problem. Discoveries made from proteomicbased studies contribute to the growing need for innovative medical diagnostics for disease detection. Taking into consideration the global health burden of cardiac disease, clinical proteomics is a valuable tool to improve risk stratification associated with this disease. In cardiovascular medicine, the identification of novel proteins, or biomarkers, that are differentially expressed in cardiac disease proteomes may enable early detection of the disease state, thereby preventing progression to disease end points. This review outlines various proteomic platforms and their technical advancements and relates these to the cardiovascular sciences. The entire protein complement of the cell, or proteome, is dynamic and changes in response to the disease state. 1 Proteomic-based experiments can be used to characterize such alterations in protein expression during disease progression. 2 With combined improvements in mass spectrometry (MS) technology as well as innovative molecular biology screening tools, there has been widespread growth in the characterization of cardiac disease proteomes. In fact, proteomic technology has been an important tool in the analysis of heart failure (HF), 3,4 cardiac hypertrophy, 5,6 and dilated cardiomyopathy.7 Several of the overarching aims of such studies include providing greater understanding of general biological mechanisms as well as identifying unique proteins that are clinically useful in the detection of cardiac disease in the early stages or potentially used as novel therapeutic targets. Clinical proteomics continues to benefit from advancements in technologies that allow for fast and consistent identification of proteins with corresponding increases in the dynamic range of proteins detectable in the disease proteome. MS-related technologies have improved in their ability to detect low-abundance proteins as well as membrane proteins and have benefited from sample preprocessing strategies that decrease the complexity of large-scale analyses. With the emergence of different methodologies for biomarker identification, the following 3 criteria must be taken into careful consideration to determine whether the protein is, in fact,

Membrane bound vesicles, including microvesicles and exosomes, are secreted by both normal and cancerous cells into the extracellular space and in blood circulation. These circulating extracellular vesicles (cirEVs) and exosomes in particular are recognized as a potential source of disease biomarkers. However, to exploit the use of circulatory exosomes as a biomarker, a rapid, high-throughput and reproducible method is required for their isolation and molecular analysis. We have developed a simple, low cost microfluidic-based platform to isolate cirEVs enriched in exosomes directly from blood serum allowing simultaneous capture and quantification of exosomes in a single device. To capture specific exosomes, we employed "ExoChip", a microfluidic device fabricated in polydimethylsiloxane (PDMS) and functionalized with antibodies against CD63, an antigen commonly overexpressed in exosomes. Subsequent staining with a fluorescent carbocyanine dye (DiO) that specifically labels the exosomes, we quantitated exosomes using a standard plate-reader. Ten independent ExoChip experiments performed using serum obtained from five pancreatic cancer patients and five healthy individuals revealed a statistically significant increase (2.34 ± 0.31 fold, p < 0.001) in exosomes captured in cancer patients when compared to healthy individuals. Exosomal origins of ExoChip immobilized vesicles were further confirmed using immuno-electron-microscopy and Western blotting. In addition, we demonstrate the ability of ExoChip to recover exosomes with intact RNA enabling profiling of exosomal-microRNAs through openarray analysis, which has potential applications in biomarker discovery. Based on our findings, ExoChip is a well suited platform to be used as an exosome-based diagnostic and research tool for molecular screening of human cancers.

Membrane bound extracellular vesicles, including microvesicles and exosomes, are secreted by both normal and cancerous cells into the extracellular space and in blood circulation. These circulating extracellular vesicles (cirEVs) and exosomes in particular, are recognized as a potential source of disease biomarkers and also carry signature molecules for early stage cancer detection. However, to exploit the use of circulatory exosomes as a new diagnostic tool, a rapid, high-throughput and reproducible method is required for their isolation and to allow their molecular analysis. We have developed a simple and a low cost microfluidic-based platform to analyze cirEVs enriched in exosomes directly from blood serum in a single step allowing simultaneous capture and quantification of exosomes. To capture exosomes with specificity, we employed a microfluidic device “ExoChip” functionalized with antibodies against exosomes to capture cirEVs. Subsequent staining with a fluorescent dye that specifically labels the exosomes, we quantitated exosomes using a standard plate-reader. Ten independent ExoChip experiments performed using serum obtained from five pancreatic cancer patients and five healthy individuals revealed a statistically significant increase (2.34±0.31 fold, p &amp;lt;0.001) in exosomes captured in cancer patients when compared to healthy individuals. Exosomal origin of ExoChip immobilized vesicles were further confirmed using immuno-electron-microscopy and Western blotting. In addition, we demonstrate the ability of ExoChip to recover exosomes with intact RNA enabling profiling of exosomal-microRNAs through openarray analysis, which has potential applications in biomarker discovery. Based on our findings, ExoChip is a well suited platform to be used as an exosome-based diagnostic and research tool for molecular screening of human cancers. Citation Format: Shailender S. Kanwar, Christopher J. Dunlay, Diane M. Simeone, Sunitha Nagrath. Simultaneous isolation and quantification of circulating exosomes for cancer biomarker discovery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5591. doi:10.1158/1538-7445.AM2014-5591

Progress in Mass Spectrometry Acquisition Approach for Quantitative Proteomics

Application of Fourier-transform ion cyclotron resonance mass spectrometry to metabolic profiling and metabolite identification

BackgroundThe world has witnessed a steady rise in neurological diseases, which represent a heterogeneous group of disorders characterized by complex pathogenesis involving disruptions at multiple molecular levels, including genomic, transcriptomic, proteomic, and metabolomic levels. These disorders, often caused by genetic mutations, metabolic imbalances, immune dysregulation, and environmental factors, pose significant challenges to global public health due to their high prevalence, mortality, and disability burden.ResultsThe advent of high-throughput technologies, such as next-generation sequencing and mass spectrometry, has provided valuable insights into the underlying mechanisms of disease, especially the development of multi- and high-spatial-resolution omics technologies, enabling the interaction of multiple levels of biology and analysis of the complex molecular networks and pathophysiological processes.ConclusionsThis review provides a comprehensive analysis of the latest advancements in multi- and high-spatial-resolution omics, with a focus on their applications in precision diagnostics, biomarker discovery, and therapeutic target identification in brain diseases. The study also highlights the current challenges in the clinical implementation and discusses the future directions, with artificial intelligence being anticipated to enhance clinical translation and diagnostic accuracy significantly.

Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and clinical challenges underlying this approach will also be discussed.

Analysis of the blood proteome allows identification of proteins related to changes upon certain physiological conditions. The pathophysiology of necrotic enteritis (NE) has been extensively studied. While intestinal changes have been very well documented, data addressing NE-induced alterations in the blood proteome are scant, although these might have merit in diagnostics. In light of recent technological advancements in proteomics and pressing need for tools to access gut health, the current study employs mass-spectrometry (MS)-based proteomics to identify biomarkers for gastrointestinal health of chickens. Here, we report findings of an untargeted proteomics investigation conducted on blood plasma in chickens under NE challenge. Two MS-strategies were used for analysis: conventional data dependent acquisition coupled to standard nanoflow liquid chromatography (LC) (nano-DDA) and recently-developed data independent acquisition coupled to an Evosep One LC system (Evo-DIA). Despite superior completeness and quantification of the Evo-DIA-acquired data, high degree of agreement in identification and quantification was observed between both approaches. Additionally, we identified 15 differentially expressed proteins (shared by nano-DDA and Evo-DIA) that represent responses of animals to infection and may serve as potential biomarkers. Experimental validation through ELISA immunoassays and targeted MS for selected regulated proteins (CFD, HPS5, and MASP2) confirmed medium-to-high levels of inter-protein correlation. A GSEA analysis revealed enrichment in a number of processes related to adaptive and humoral immunity, immune activation and response in infected animals. Data are available via ProteomeXchange with identifiers PXD050461, PXD050473, and PXD061607.

Mass spectrometry technology is becoming an important tool for clinical analysis due to its high specificity, high sensitivity and high multi-component detection capability. The current applications of this technology are mainly in liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF-MS), inductively coupled plasma mass spectrometry (ICP-MS), gas chromatography-mass spectrometry (GC-MS) and the related in vitro diagnostic kits. At present, the number of medical device (MD) based on mass spectrometry technology is growing rapidly, especially the number of LC-MS/MS and MALDI-TOF-MS registered MD products, and the standardization of relevant product quality requirements is also being effectively carried out. In general, clinical mass spectrometry equipment is still mainly imported, and the equipment price is relatively high. The development of mass spectrometry kits is mainly based on imported platforms, and domestic equipment is still in its infancy; the further promotion of clinical application of mass spectrometry also depends on the progress of the automation and standardization of the analysis procedure. To investigate the detection performance of mass spectrometry systems, it is necessary to fully consider the characteristics of mass spectrometry technology itself.

In the era of quantitative proteomics, where mass spectrometry plays a pivotal role, in particular associated with the use of data-independent acquisition, it is time to perform an overview of this growing field with special focus on one of the most promising approaches: SWATH-MS, and to present future perspectives for its application as a translational tool. Therefore, a summary of this technique is presented focusing on two key relevant concepts associated with its application in biomarker discovery: the protein library and the individual digital maps concepts. It is also the purpose of this review to document the likely impact of SWATH-MS in both fundamental and translational research including biomarker identification and creation of diagnostic tools. To that end, the two concepts referred above were integrated with ongoing technical developments. Finally, some of the current restrictions for the implementation of SWATH-MS on a large scale are identified, and potential solutions presented, namely protocol standardization combined with the use of the proper standards.