Integral membrane proteins: bottom-up,top-down and structural proteomics

Introduction: Integral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review.

Areas covered: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing critical in-vivo information.

Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized.     

Post-translational quantitation by SRM/MRM: applications in cardiology

Introduction: Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization, and interaction with other molecules. PTMs apply a dynamic control of proteins in both physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution mass spectrometry (MS)/MS analysis provide attractive results on PTM characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples.

Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported, and a specific section is focused on PTM detection in proteins that are involved in the cardiovascular system and heart diseases.

Expert commentary: PTM characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded our knowledge in the last few years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTM complexity. Promising studies confirm the great potential of SRM/MRM to study PTMs in the cardiovascular field, and PTMomics could be very useful in the clinical perspective.     

Molecular insights into cancer drug resistance from a proteomics perspective

Introduction: Resistance to chemotherapy and development of specific and effective molecular targeted therapies are major obstacles facing current cancer treatment. Comparative proteomic approaches have been employed for the discovery of putative biomarkers associated with cancer drug resistance and have yielded a number of candidate proteins, showing great promise for both novel drug target identification and personalized medicine for the treatment of drug-resistant cancer.

Areas covered: Herein, we review the recent advances and challenges in proteomics studies on cancer drug resistance with an emphasis on biomarker discovery, as well as understanding the interconnectivity of proteins in disease-related signaling pathways. In addition, we highlight the critical role that post-translational modifications (PTMs) play in the mechanisms of cancer drug resistance.

Expert opinion: Revealing changes in proteome profiles and the role of PTMs in drug-resistant cancer is key to deciphering the mechanisms of treatment resistance. With the development of sensitive and specific mass spectrometry (MS)-based proteomics and related technologies, it is now possible to investigate in depth potential biomarkers and the molecular mechanisms of cancer drug resistance, assisting the development of individualized therapeutic strategies for cancer patients.     

Recent insights into human bronchial proteomics – how are we progressing and what is next?

Introduction: The human respiratory system is highly prone to diseases and complications. Many lung diseases, including lung cancer (LC), tuberculosis (TB), and chronic obstructive pulmonary disease (COPD) have been among the most common causes of death worldwide. Cystic fibrosis (CF), the most common genetic disease in Caucasians, has adverse impacts on the lungs. Bronchial proteomics plays a significant role in understanding the underlying mechanisms and pathogenicity of lung diseases and provides insights for biomarker and therapeutic target discoveries.

Areas covered: We overview the recent achievements and discoveries in human bronchial proteomics by outlining how some of the different proteomic techniques/strategies are developed and applied in LC, TB, COPD, and CF. Also, the future roles of bronchial proteomics in predictive proteomics and precision medicine are discussed.

Expert commentary: Much progress has been made in bronchial proteomics. Owing to the advances in proteomics, we now have better ability to isolate proteins from desired cellular compartments, greater protein separation methods, more powerful protein detection technologies, and more sophisticated bioinformatic techniques. These all contributed to our further understanding of lung diseases and for biomarker and therapeutic target discoveries.     

Proteomic approaches for cancer epigenetics research

Introduction: Epigenetic dysregulation drives or supports numerous human cancers. The chromatin landscape in cancer cells is often marked by abnormal histone post-translational modification (PTM) patterns and by aberrant assembly and recruitment of protein complexes to specific genomic loci. Mass spectrometry-based proteomic analyses can support the discovery and characterization of both phenomena.

Areas covered: We broadly divide this literature into two parts: ‘modification-centric’ analyses that link histone PTMs to cancer biology; and ‘complex-centric’ analyses that examine protein–protein interactions that occur de novo as a result of oncogenic mutations. We also discuss proteomic studies of oncohistones. We highlight relevant examples, discuss limitations, and speculate about forthcoming innovations regarding each application.

Expert commentary: ‘Modification-centric’ analyses have been used to further understanding of cancer’s histone code and to identify associated therapeutic vulnerabilities. ‘Complex-centric’ analyses have likewise revealed insights into mechanisms of oncogenesis and suggested potential therapeutic targets, particularly in MLL-associated leukemia. Proteomic experiments have also supported some of the pioneering studies of oncohistone-mediated tumorigenesis. Additional applications of proteomics that may benefit cancer epigenetics research include middle-down and top-down histone PTM analysis, chromatin reader profiling, and genomic locus-specific protein identification. In the coming years, proteomic approaches will remain powerful ways to interrogate the biology of cancer.     

Mass spectrometry-based proteomic exploration of the human immune system: focus on the inflammasome,global protein secretion,and T cells

Introduction: The immune system is our defense system against microbial infections and tissue injury, and understanding how it works in detail is essential for developing drugs for different diseases. Mass spectrometry-based proteomics can provide in-depth information on the molecular mechanisms involved in immune responses.

Areas covered: Summarized are the key immunology findings obtained with MS-based proteomics in the past five years, with a focus on inflammasome activation, global protein secretion, mucosal immunology, immunopeptidome and T cells. Special focus is on extracellular vesicle-mediated protein secretion and its role in immune responses.

Expert commentary: Proteomics is an essential part of modern omics-scale immunology research. To date, MS-based proteomics has been used in immunology to study protein expression levels, their subcellular localization, secretion, post-translational modifications, and interactions in immune cells upon activation by different stimuli. These studies have made major contributions to understanding the molecular mechanisms involved in innate and adaptive immune responses. New developments in proteomics offer constantly novel possibilities for exploring the immune system. Examples of these techniques include mass cytometry and different MS-based imaging approaches which can be widely used in immunology.     

Middle-down proteomics: a still unexploited resource for chromatin biology

Introduction: Analysis of histone post-translational modifications (PTMs) by mass spectrometry (MS) has become a fundamental tool for the characterization of chromatin composition and dynamics. Histone PTMs benchmark several biological states of chromatin, including regions of active enhancers, active/repressed gene promoters and damaged DNA. These complex regulatory mechanisms are often defined by combinatorial histone PTMs; for instance, active enhancers are commonly occupied by both marks H3K4me1 and H3K27ac. The traditional bottom-up MS strategy identifies and quantifies short (aa 4–20) tryptic peptides, and it is thus not suitable for the characterization of combinatorial PTMs.

Areas covered: Here, we review the advancement of the middle-down MS strategy applied to histones, which consists in the analysis of intact histone N-terminal tails (aa 50–60). Middle-down MS has reached sufficient robustness and reliability, and it is far less technically challenging than PTM quantification on intact histones (top-down). However, the very few chromatin biology studies applying middle-down MS resulting from PubMed searches indicate that it is still very scarcely exploited, potentially due to the apparent high complexity of method and analysis.

Expert commentary: We will discuss the state-of-the-art workflow and examples of existing studies, aiming to highlight its potential and feasibility for studies of cell biologists interested in chromatin and epigenetics.     

Tissue proteomics studies in the investigation of prostate cancer

Introduction: Prostate cancer (PCa) is one of the leading causes of death in the male population worldwide. Various clinical samples such as urine, blood serum, and prostatic fluid have been commonly used for the identification of PCa-associated molecular changes. Tissue, the site of oncogenesis, is increasingly gaining more attention as a study material for studies aimed at the discovery of biomarkers for predicting the disease outcome and therapeutic targets.

Areas covered: This review is the output of a systematic literature search on PubMed to retrieve articles relevant to the proteomic analysis of tissues for the study of PCa. Studies performed during the last 10 years using human tissues are summarized.

Expert commentary: Multiple proteomics studies were performed in the past 10 years focusing on PCa initial diagnosis and staging. Even though some reproducible findings have been reported, many studies lacked adequate validation of findings and relied on relatively lower-resolution proteomics techniques compared to the current state of the art. Incorporation of high-resolution proteomics techniques, including investigations of protein post-translational modifications (PTMs), is expected in the near future to complement other -omics and enhance current efforts toward the molecular subtyping of PCa for patient stratification.     

Proteomics landscape of radiation-induced cardiovascular disease: somewhere over the paradigm

Introduction: Epidemiological studies clearly show that thoracic or whole body exposure to ionizing radiation increases the risk of cardiac morbidity and mortality. Radiation-induced cardiovascular disease (CVD) has been intensively studied during the last ten years but the underlying molecular mechanisms are still poorly understood.

Areas covered: Heart proteomics is a powerful tool holding promise for the future research. The central focus of this review is to compare proteomics data on radiation-induced CVD with data arising from proteomics of healthy and diseased cardiac tissue in general. In this context we highlight common and unique features of radiation-related and other heart pathologies. Future prospects and challenges of the field are discussed.

Expert commentary: Data from comprehensive cardiac proteomics have deepened the knowledge of molecular mechanisms involved in radiation-induced cardiac dysfunction. State-of-the-art proteomics has the potential to identify novel diagnostic and therapeutic markers of this disease.     

A systems biology-led insight into the role of the proteome in neurodegenerative diseases

Introduction: Multifactorial disorders are the result of nonlinear interactions of several factors; therefore, a reductionist approach does not appear to be appropriate. Proteomics is a global approach that can be efficiently used to investigate pathogenetic mechanisms of neurodegenerative diseases.

Areas covered: Here, we report a general introduction about the systems biology approach and mechanistic insights recently obtained by over-representation analysis of proteomics data of cellular and animal models of Alzheimer’s disease, Parkinson’s disease and other neurodegenerative disorders, as well as of affected human tissues.

Expert commentary: As an inductive method, proteomics is based on unbiased observations that further require validation of generated hypotheses. Pathway databases and over-representation analysis tools allow researchers to assign an expectation value to pathogenetic mechanisms linked to neurodegenerative diseases. The systems biology approach based on omics data may be the key to unravel the complex mechanisms underlying neurodegeneration.     

Proteomic analysis of mitochondria: biological and clinical progresses in cancer

Introduction: Mitochondria play important roles in regulating multiple biological processes and signalling pathways in eukaryotic cells, and mitochondrial dysfunction may result in a wide range of serious diseases, including cancer. With improvements in the identification of mitochondrial proteins, mitochondrial proteomics has made great achievements. In particular, this approach has been widely used to compare tumour cells at different stages of malignancy. Therefore, there is an urgent need to identify and characterize the function of mitochondrial proteins in cancer progression and to determine the involved mechanisms.

Areas covered: We provide an overview of recent progress related to mitochondrial proteomics in cancer and the application of comparative mitochondrial proteomics in various biological processes, including apoptosis, necroptosis, autophagy and metastasis, as well as clinical progress in cancer. Proteomics-related reports were found using PubMed and Google Scholar databases.

Expert commentary: Understanding both post-translational modification and post-translational processing is important in the comprehensive characterization of protein function. The application of comparative mitochondrial proteomics to investigate clinical samples and cancer cells will contribute to our understanding of the molecular interplay of mitochondrial proteins in the development of cancer. This approach will mine more biomarkers for diagnosis and prognosis and improve therapeutic outcomes among cancer patients.     

Applying proteomics to tick vaccine development: where are we?

Introduction: Ticks are second to mosquitoes as a vector of human diseases and are the first vector of animal diseases with a great impact on livestock farming. Tick vaccines represent a sustainable and effective alternative to chemical acaricides for the control of tick infestations and transmitted pathogens. The application of proteomics to tick vaccine development is a fairly recent area, which has resulted in the characterization of some tick-host-pathogen interactions and the identification of candidate protective antigens.

Areas covered: In this article, we review the application and possibilities of various proteomic approaches for the discovery of tick and pathogen derived protective antigens, and the design of effective vaccines for the control of tick infestations and pathogen infection and transmission.

Expert commentary: In the near future, the application of reverse proteomics, immunoproteomics, structural proteomics, and interactomics among other proteomics approaches will likely contribute to improve vaccine design to control multiple tick species with the ultimate goal of controlling tick-borne diseases.     

The application of gene silencing in proteomics: from laboratory to clinic

Introduction: Since the completion of genome sequencing, gene silencing technologies have emerged as powerful tools to study gene functions in various biological processes, both in vivo and in vitro. Moreover, they have also been proposed as therapeutic agents to inhibit selected genes in a variety of pathological conditions, such as cancer, neurodegenerative, and cardiovascular diseases.

Area covered: This review summarizes the mechanisms of action and applications of genome editing tools, from RNA interference to clustered regularly interspaced short palindromic repeats-based systems, in research and in clinics. We describe their essential role in high-throughput genetic screens and, in particular, in functional proteomics studies, to identify diagnostic markers and therapeutic targets. Indeed, gene silencing and proteomics have been extensively integrated to study global proteome changes, posttranslational modifications, and protein–protein interactions.

Expert commentary: Functional proteomics approaches that leverage gene silencing tools have been successfully applied to examine the role of several genes in various contexts, leading to a deeper knowledge of biological pathways and disease mechanisms. Recent developments of gene silencing tools have improved their performance, also in terms of off-targets effects reduction, paving the way for a wider therapeutic application of these systems.     

Proteomics of Pseudomonas aeruginosa: the increasing role of post-translational modifications

Introduction: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen widely involved in human infections. The high occurrence of this bacterial species in the clinical field is due to its high ability to adapt to detrimental environments, in particular its strong inherent antibiotic resistance, its ability to form biofilms and to produce virulence factors. The application of proteomics to clinical microbiology is probably one of the most innovative strategies of the last decades to understand complex microbial systems, by providing individual proteome charts of pathogens.

Areas covered: In the last decade, proteomic advances have allowed in high-throughput the screening of proteins modified by diverse co- and post-translational modifications in P. aeruginosa. This review will present the current state of the art for the characterization of PTMs in P. aeruginosa by proteomics approaches. We will then discuss on the involvement of PTMs in P. aeruginosa physiology.

Expert commentary: Modified proteins and enzymes involved in the addition/removal of modifications will surely constitute targets of interest to develop new therapeutic drugs to fight against P. aeruginosa.     

Advances in proteomic study of cardiac amyloidosis: progress and potential

Introduction: More than ten distinct forms of amyloidoses that can involve the heart have been described, classified according to which protein originates the deposits. Cardiac amyloid infiltration translates into progressive and often life-threatening cardiomyopathy, but disease severity, prognosis and treatment drastically differ according to the amyloidosis type. The notion that protein misfolding and aggregation play a more general role in human cardiomyopathies has further raised attention towards the definition of the proteotoxicity mechanisms.

Areas covered: Mass spectrometry-based proteomics plays an important role as a diagnostic tool and for understanding the molecular bases of amyloid cardiomyopathies. The landscape of applications of proteomics to the study of cardiac amyloidoses and amyloid-related cardiotoxicity is summarized, with a critical synthesis of the major achievements.

Expert commentary: Current strengths and limitations of proteomics in the clinical setting and in translational research on amyloid cardiomyopathy are discussed, with the foreseen potential future directions in the field.     

Snake venomics – from low-resolution toxin-pattern recognition to toxin-resolved venom proteomes with absolute quantification

Introduction: Venoms are integrated phenotypes used by a wide range of organisms for predatory and defensive purposes. The study of venoms is of great interest in diverse fields, such as evolutionary ecology and biotechnology. Omics technologies have contributed to understanding the evolutionary mechanisms that molded snake venoms to their present-day structural and functional variability landscape.

Areas covered: This review article reflects on two recent implementations in venomics: absolute quantification of intact proteins by elemental mass spectrometry, and top-down molecular mass spectrometry.

Expert commentary: Leveraging on a new way of polyatomic interference removal, a triple quadrupole inductively coupled plasma mass spectrometry configuration has proven feasible for the absolute quantification of venom toxins via sulfur detection. A major advantage of this approach over quantitative molecular mass spectrometry techniques is that only a generic S-standard is required to quantify all the chromatographically separated sulfur-containing fractions. Top-down venomics is in its infancy but, due to recent hardware and software developments, is gaining momentum. Proteoform-resolved venom proteomes are needed to understand the spatio-temporal variability landscape underlying the adaptations that drive intraspecific venom evolution. Integrating top-down venomics and absolute proteoform quantification into a novel elemental and molecular mass spectrometry configuration will represent a quantitative leap in the study of individual venoms.