Expert Review of Proteomics最新文献

Introduction: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times.

Areas covered: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant.

Expert opinion: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.

Introduction: Investigating the taxonomic and functional composition of human microbiomes can aid in the understanding of disease etiologies, diagnosis, and therapy monitoring for several diseases, including inflammatory bowel disease or obesity. One method for microbiome monitoring is metaproteomics, which assesses human and microbial proteins and thus enables the study of host-microbiome interactions. This advantage led to increased interest in metaproteome analyses and significant developments to introduce this method into a clinical context.

Areas covered: This review summarizes the recent progress from a technical side and an application-related point of view.

Expert opinion: Numerous publications imply the massive potential of metaproteomics to impact human health care. However, the key challenges of standardization and validation of experimental and bioinformatic workflows and accurate quantification methods must be overcome.

Introduction: Intrinsically disordered proteins (IDPs) represent a family of proteins that lack secondary or tertiary structure. IDPs are hubs in interaction networks, participate in liquid-liquid phase separation processes, and drive the formation of proteinaceous membrane-less organelles. Their unfolded structure makes them particularly prone to post-translational modifications (PTMs) that play key functional modulatory roles.

Areas covered: We discuss different analytical approaches to study phosphorylation of IDPs starting from methods for IDP enrichment (strong acid extractions and heat-based pre-fractionation), strategies to enrich and map phosphopeptides/proteins, and mass spectrometry-based tools to study the phosphorylation-dependent conformational alterations of IDPs (limited proteolysis, HDX, chemical cross-linking, covalent labeling, and ion mobility).

Expert opinion: There is a growing interest in IDPs and their PTMs since they are involved in several diseases. The intrinsic disorder could be exploited to facilitate purification and synthetic production of IDPs taking full advantage of those structural mass-spectrometry-based methods that can be used to investigate IDPs and their phospho-dependent conformational alterations. The diffusion and implementation of mass spectrometers with ion mobility devices and electron transfer dissociation capabilities could be key-elements for increasing information on IDP biology.

Objectives: Hereditary transthyretin amyloidosis (ATTRv) is a rare, fatal, autosomal dominant disease with more than 140 mutations discovered. Three phenotypes of amyloid infiltration are neuropathy (ATTRv-PN), cardiopathy (ATTRv-CM), and neuropathy + cardiopathy (ATTRv-MIX). The lack of ATTR-specific biomarkers, difficulties in biopsy evidence, and limited known pathogenic mechanisms have made diagnosis difficult. Newly emerging noninvasive measures for monitoring progression and disease-modifying therapies have improved early diagnosis and patient management.

Methods: Our research applies the latest technology, Data-Independent Acquisition-Based Quantitative Proteomics (DIA), to reveal comprehensive plasma protein profiles in the natural history of Chinese patients with hereditary transthyretin amyloidosis (ATTRv). We analyzed differentially expressed proteins (DEPs) in three phenotypes (ATTRv-PN, ATTRv-CM, and ATTRv-MIX).

Results: Serum samples were collected from a total of 18 patients (6 ATTRv-PN, 5 ATTRv-CM, and 7 ATTRv-MIX patients) and 20 healthy participants as a control group. Combined with the results of the proteomic and bioinformatic analyses, we found 30 DEPs and protein interaction networks clustered in KRT family proteins and DSC3 between ATTRv-PN and the control, which were rich in the estrogen signaling pathway and the cell adhesion molecule (CAM) pathway.

Conclusion: This study demonstrates a global and significant proteomic profile in different stages of ATTRv.

Introduction: The COVID-19 outbreak has put enormous pressure on the scientific community to detect infection rapidly, identify the status of disease severity, and provide an immediate vaccine/drug for the treatment. Relying on immunoassay and a real-time reverse transcription polymerase chain reaction (rRT-PCR) led to many false-negative and false-positive reports. Therefore, detecting biomarkers is an alternative and reliable approach for determining the infection, its severity, and disease progression. Recent advances in liquid chromatography and mass spectrometry (LC-MS/MS) enable the protein biomarkers even at low concentrations, thus facilitating clinicians to monitor the treatment in hospitals.

Areas covered: This review highlights the role of LC-MS/MS in identifying protein biomarkers and discusses the clinically significant protein biomarkers such as Serum amyloid A, Interleukin-6, C-Reactive Protein, Lactate dehydrogenase, D-dimer, cardiac troponin, ferritin, Alanine transaminase, Aspartate transaminase, gelsolin and galectin-3-binding protein in COVID-19, and their analysis by LC-MS/MS in the early stage.

Expert opinion: Clinical doctors monitor significant biomarkers to understand, stratify, and treat patients according to disease severity. Knowledge of clinically significant COVID-19 protein biomarkers is critical not only for COVID-19 caused by the coronavirus but also to prepare us for future pandemics of other diseases in detecting by LC-MS/MS at the early stages.

Introduction: Spermatozoa are highly specialized cells with unique morphology. In addition, spermatozoa lose a considerable amount of cytoplasm during spermiogenesis, when they also compact their DNA, resulting in a transcriptionally quiescent cell. Throughout the male reproductive tract, sperm will acquire proteins that enable them to interact with the female reproductive tract. After ejaculation, proteins undergo post-translational modifications for sperm to capacitate, hyperactivate, and fertilize the oocyte. Many proteins have been identified as predictors of male infertility and also investigated in diseases that compromise reproductive potential.

Areas covered: In this review, we proposed to summarize the recent findings about the sperm proteome and how they affect sperm structure, function, and fertility. A literature search was performed using PubMed and Google Scholar databases within the past 5 years until August 2022.

Expert opinion: Sperm function depends on protein abundance, conformation, and PTMs; understanding the sperm proteome may help to identify pathways essential to fertility, even making it possible to unravel the mechanisms involved in idiopathic infertility. In addition, proteomics evaluation offers knowledge regarding alterations that compromise the male reproductive potential.

Introduction: The lysosome is the main degradative organelle of almost all mammalian cells, fulfilling important functions in macromolecule recycling, metabolism, and signaling. Lysosomal dysfunction is connected to a continuously growing number of pathologic conditions, and lysosomal proteins present potential biomarkers for a variety of diseases. Therefore, there is an increasing interest in their analysis in patient samples.

Areas covered: We provide an overview of OMICs studies which identified lysosomal proteins as potential biomarkers for pathological conditions, covering proteomics, genomics, and transcriptomics approaches, identified through PubMed searches. With respect to discovery proteomics analyses, mainly lysosomal luminal and associated proteins were detected, while membrane proteins were found less frequently. Comprehensive coverage of the lysosomal proteome was only achieved by ultra-deep-coverage studies, but targeted approaches allowed for the reproducible quantification of lysosomal proteins in diverse sample types.

Expert opinion: The low abundance of lysosomal proteins complicates their reproducible analysis in patient samples. Whole proteome shotgun analyses fail in many instances to cover the lysosomal proteome, which is due to under-sampling and/or a lack of sensitivity. With the current state of the art, targeted proteomics assays provide the best performance for the characterization of lysosomal proteins in patient samples.

Introduction: The creation of ProteomeXchange data workflows in 2012 transformed the field of proteomics, consisting of the standardization of data submission and dissemination and enabling the widespread reanalysis of public MS proteomics data worldwide. ProteomeXchange has triggered a growing trend toward public dissemination of proteomics data, facilitating the assessment, reuse, comparative analyses, and extraction of new findings from public datasets. By 2022, the consortium is integrated by PRIDE, PeptideAtlas, MassIVE, jPOST, iProX, and Panorama Public.

Areas covered: Here, we review and discuss the current ecosystem of resources, guidelines, and file formats for proteomics data dissemination and reanalysis. Special attention is drawn to new exciting quantitative and post-translational modification-oriented resources. The challenges and future directions on data depositions including the lack of metadata and cloud-based and high-performance software solutions for fast and reproducible reanalysis of the available data are discussed.

Expert opinion: The success of ProteomeXchange and the amount of proteomics data available in the public domain have triggered the creation and/or growth of other protein knowledgebase resources. Data reuse is a leading, active, and evolving field; supporting the creation of new formats, tools, and workflows to rediscover and reshape the public proteomics data.

Introduction: The peritoneum, pleura, and pericardium are yet understudied multicellular systems where mesothelial cells (MCs) and endothelial cells (ECs) are in close proximity. Crosstalk between these cell types likely plays role in molecular transport, immunological reactions, and metabolic processes in health, disease, and therapeutic intervention.

Areas covered: In this review, we discuss recent proteomic efforts to characterize the crosstalk between MC and EC. We describe the proteomic methods necessary for investigation of crosstalk between MC and EC, as well as the in-vitro models that can be employed. Potential experimental approaches range from conditioned medium, via co-culture on semi-permeable membranes, to 3D cell culture based organoid models. While the biological and clinical relevance of the models may increase with their ability to mimic close cell communication, the practicality of these complex experiments corresponds vice versa, making standardization more difficult and expensive.

Expert opinion: Currently, data and reports on mesothelial-to-endothelial crosstalk are still very scarce. In our opinion, the in-vitro model using semi-permeable cell culture inserts will allow to establish a basic understanding of cellular crosstalk that may occur between those cell types. Later-on, more sophisticated 3D cell cultures may be better able to simulate the transport dynamics within the peritoneal membrane.