Expert Review of Proteomics最新文献

Background: Botulinum neurotoxins (BoNTs) are a group of neurotoxins produced by Clostridium bacteria. Their effect on neuro-muscular connections through cleaving proteins of the SNARE complex results in blocking acetylcholine signal transduction. The FDA-approved mouse bioassay, which involves exposing live mice to potentially contaminated food, is the most widely used method. However, this assay is costly, time-consuming, and raises ethical concerns. Therefore, there is a need for alternative assays that can enzymatically measure the activity of BoNTs.

Research design and methods: We present an approach that combines the EndoPep-MS assay with protein affinity chips fabricated using ion soft-landing technology. Toxic activity is indirectly assessed by monitoring the N- and C-terminal fragments of the substrate peptide. This new method employs a protein array with affinity molecules targeting either the BoNT/A1 or the substrate peptide. Both variants enable in-situ reaction and detection of substrate peptides via MALDI-ToF MS on the protein chip.

Results: This method demonstrated successful detection of active BoNT/A1 in both buffer and complex matrices, achieving a detection limit of 0.5 ng/mL.

Conclusions: This study reports the in-situ detection of botulotoxin A1 using functionalized MALDI chips. The advantages of the MALDI chip technology include speed, robustness, cost-effectiveness, and possible automatization.

Introduction: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been a leading method for proteomics for thirty years. Advantages provided by LC-MS/MS are offset by significant disadvantages, including cost. Recently, several non-mass spectrometric methods have emerged, but little information is available about their capacity to analyze the complex mixtures routine for mass spectrometry.

Areas covered: We review recent non-mass-spectrometric methods for sequencing proteins and peptides, including using nanopores, sequencing by degradation, reverse translation, and short-epitope mapping, with comments on bioinformatics challenges, fundamental limitations, and areas where new technologies will be more or less competitive with LC-MS/MS. In addition to conventional literature searches, instrument vendor websites, patents, webinars, and preprints were also consulted to give a more up-to-date picture.

Expert opinion: Many new technologies are promising. However, demonstrations that they outperform mass spectrometry in terms of peptides and proteins identified have not yet been published, and astute observers note important disadvantages, especially relating to the dynamic range of single-molecule measurements of complex mixtures. Still, even if the performance of emerging methods proves inferior to LC-MS/MS, their low cost could create a different kind of revolution: a dramatic increase in the number of biology laboratories engaging in new forms of proteomics research.

Introduction: Ligand binding assays combining immunoaffinity enrichment steps with mass spectrometry (MS) readout have gained attention as a highly specific and sensitive tool for protein quantification. These techniques typically combine enzymatic fragmentation of the sample or enriched protein with capture on the protein or peptide-level for quantification. Antibodies ensure specific target recognition, while MS offers quantitative accuracy with isotopically labeled internal standards. This dual approach supports a broad dynamic range, enabling protein measurements from picomolar to nanomolar levels. These methods have diverse applications, from quantifying signaling proteins in basic research to biomarker monitoring in clinical trials and analyzing the pharmacokinetics of therapeutic proteins.

Areas covered: This review delves into the diverse workflows of immunoaffinity-MS, shedding light on the innovative strategies employed, their practical applications, efficacy, and inherent limitations in the realm of protein quantification.

Expert opinion: Immunoaffinity-MS has transformed protein analysis, but widespread adoption is hindered by complex workflows, high instrument costs, and limited capture molecule availability. Efforts to enhance automation, standardize workflows, and advance technological innovation aim to overcome these barriers. Improvements in mass spectrometer sensitivity, advances in recombinant capture technologies, and support from public initiatives are poised to further improve the reliability and accessibility of this method.

Introduction: Rare diseases (RDs) are a heterogeneous group of diseases recognized as a relevant global health priority but posing aspects of complexity, such as geographical scattering of affected individuals, improper/late diagnosis, limited awareness, difficult surveillance and monitoring, limited understanding of natural history, and lack of treatment. Usually, RDs have a pediatric onset and are life-long, multisystemic, and associated with a poor prognosis.

Areas covered: In this work, we review how high-throughput omics technologies such as genomics, transcriptomics, proteomics, metabolomics, epigenomics, and other well-established omics, which are increasingly more affordable and efficient, can be applied to the study of RDs promoting diagnosis, understanding of pathological mechanisms, biomarker discovery, and identification of treatments.

Expert opinion: RDs, despite their challenges, offer a niche where collaborative efforts and personalized treatment strategies might be feasible using omics technologies. Specialized consortia fostering multidisciplinary collaboration, data sharing, and the development of biobanks and registries can be built; multi-omics approaches, including so far less exploited omics technologies, along with the implementation of AI tools can be undertaken to deepen our understanding of RDs, driving biomarker discovery and clinical interventions. Nevertheless, technical, ethical, legal, and societal issues must be clearly defined and addressed.

Introduction: The DeepMind's AlphaFold (AF) has revolutionized biomedical and biocience research by providing both experts and non-experts with an invaluable tool for predicting protein structures. However, while AF is highly effective for predicting structures of rigid and globular proteins, it is not able to fully capture the dynamics, conformational variability, and interactions of proteins with ligands and other biomacromolecules.

Areas covered: In this review, we present a comprehensive overview of the latest advancements in 3D model predictions for biomacromolecules using AF. We also provide a detailed analysis its of strengths and limitations, and explore more recent iterations, modifications, and practical applications of this strategy. Moreover, we map the path forward for expanding the landscape of AF toward predicting structures of every protein and peptide, and their interactions in the proteome in the most physiologically relevant form. This discussion is based on an extensive literature search performed using PubMed and Google Scholar.

Expert opinion: While significant progress has been made to enhance AF's modeling capabilities, we argue that a combined approach integrating both various in silico and in vitro methods will be most beneficial for the future of structural biology, bridging the gaps between static and dynamic features of proteins and their functions.

Objective: Our study presents a novel analysis of the oncogenes and tumor suppressor proteins directly modulated by E6/E7 of high-risk HPV types 16 and 18, in colorectal cancer (CRC).

Methods: HCT 116 (KRAS mutant) & HT-29 (TP53 mutant) cell models of CRC were transduced with E6/E7 of HPV16 and HPV18, individually and in combination. Further, we utilized a liquid chromatography mass spectrometry (LC-MS/MS) approach to analyze and compare the proteomes of both CRC cell models.

Results: We generated six stably transduced cell lines. Our data revealed a significantly higher, HPV-induced modulation of oncogenes and tumor suppressor proteins in the TP53 mutant model, as compared to the KRAS mutant model (p ≤ 0.01). Less than 1% of the genes were commonly modulated by HPV, between both models. We also report that HT-29 cells, expressing E6/E7 of both HPV types, significantly reduced the suppression of oncogenes as compared to cells expressing E6/E7 of either HPV types individually (p-value ≤0.00001).

Conclusion: Our data imply that HPV coinfections leads to the sustenance of a pro-oncogenic environment in CRC. HPV modulates different oncogenes/tumor suppressor proteins in CRC of varying mutational backgrounds, thus highlighting the importance of personalized therapies for such diseases with mutational heterogeneity.

Introduction: Exogenous Cushing's syndrome is the result of long-term exposure to glucocorticoids, while endogenous Cushing's syndrome occurs due to excessive production of glucocorticoids within the body. Cushing's syndrome remains a diagnostic challenge for the treating physician.Mass spectrometry, with its better resolution, detectability, and specificity, paved the way to understanding the cellular and molecular mechanisms involved in several diseases that facilitated the evolution of biomarkers and personalized medicine, which can be applicable to manage Cushing's syndrome as well.

Areas covered: There are only a few reports of mass spectrometry-based metabolomic approaches to endogenous Cushing's syndrome of certain etiologies. However, the application of this approach in the diagnosis of exogenous Cushing has not been explored much. This review attempts to discuss the application of the mass spectrometry-based metabolomic approach in the evaluation of Cushing's syndrome.

Expert opinion: Global metabolomics has the potential to discover altered metabolites and associated signaling and metabolic pathways, which may serve as potential biomarkers that would help in developing tools to accelerate precision medicine. Multi-omics approaches will provide innovative solutions to develop molecular tests for multi-molecule panel assays.

Introduction: Identifying early risks of developing Alzheimer's disease (AD) is a major challenge as the number of patients with AD steadily increases and requires innovative solutions. Current molecular diagnostic modalities, such as cerebrospinal fluid (CSF) testing and positron emission tomography (PET) imaging, exhibit limitations in their applicability for large-scale screening. In recent years, there has been a marked shift toward the development of blood plasma-based diagnostic tests, which offer a more accessible and clinically viable alternative for widespread use. Furthermore, advances in large-scale proteomics technologies have boosted an interest in identifying novel biomarkers and developing panels of AD-associated proteins.

Areas covered: This review mainly examines the results of recent searches for proteomic markers of AD in blood plasma (from 2022-2024 PubMed), focuses on some aspects for special attention in further studies, and discusses the prospects for their further application.

Expert opinion: Recent advances in AD plasma/serum proteomic studies are largely driven using novel Olink/PEA and SomaScan/aptamer technologies, which complement the 'gold standard' of MS-based quantitative proteomics (MRM/SRM), and particularly expand the capabilities for studying low-abundant proteins.

Introduction: Recent work identified members of the evolutionarily conserved coronin protein family as key regulators of cell population size. This work originated ~25 years ago through the identification, by two-dimensional gel electrophoresis, of coronin 1 as a host protein involved in the virulence of Mycobacterium tuberculosis. We here describe the journey from a spot on a 2D gel to the recent realization that coronin proteins represent key controllers of eukaryotic cell population sizes, using ever more sophisticated proteomic techniques.

Areas covered: We discuss the value of 'old school' proteomics using relatively simple and cost-effective technologies that allowed to gain insights into subcellular proteomes and describe how label-free quantitative (phospho)proteomics using mass spectrometry allowed to disentangle the role for coronin 1 in eukaryotic cell population size control. Finally, we mention potential implications of coronin-mediated cell population size control for health and disease.

Expert opinion: Proteome analysis has been revolutionized by the advent of modern-day mass spectrometers and is indispensable for a better understanding of biology. Here, we discuss how careful dissection of physio-pathological processes by a combination of proteomics, genomics, biochemistry and cell biology may allow to zoom in on the unexplored, thereby possibly tackling hitherto unasked questions and defining novel mechanisms.