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The goal of protein purification is to separate a specific protein from all other biomolecules. Classical chromatographic procedures have been designed to exploit particular distinguishing features of individual target proteins, such as size, shape, physicochemical properties, and binding affinity. Advances in molecular biology and bioinformatics have positively contributed at every level to the challenge of purifying individual proteins and more recently have led to the development of high-throughput proteomic platforms. In this chapter, a synopsis of advancements in the field of protein chromatography is presented, with reference to the principal tools and resources that are available to assist with protein purification strategies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Data from genomics, proteomics, structural biology and cryo-electron microscopy are integrated into a structural illustration of a cross section through an entire JCVI-syn3.0 minimal cell. The illustration is designed with several goals: to inspire excitement in science, to depict the underlying scientific results accurately, and to be feasible in traditional media. Design choices to achieve these goals include reduction of visual complexity with simplified representations, use of orthographic projection to retain scale relationships, and an approach to color that highlights functional compartments of the cell. Given that this simple cell provides an attractive laboratory for exploring the central processes needed for life, several functional narratives are included in the illustration, including division of the cell and the first depiction of an entire cellular proteome. The illustration lays the foundation for 3D molecular modeling of this cell., (© 2022 the author(s), published by De Gruyter, Berlin/Boston.)

The 2022 Nucleic Acids Research Database Issue contains 185 papers, including 87 papers reporting on new databases and 85 updates from resources previously published in the Issue. Thirteen additional manuscripts provide updates on databases most recently published elsewhere. Seven new databases focus specifically on COVID-19 and SARS-CoV-2, including SCoV2-MD, the first of the Issue's Breakthrough Articles. Major nucleic acid databases reporting updates include MODOMICS, JASPAR and miRTarBase. The AlphaFold Protein Structure Database, described in the second Breakthrough Article, is the stand-out in the protein section, where the Human Proteoform Atlas and GproteinDb are other notable new arrivals. Updates from DisProt, FuzDB and ELM comprehensively cover disordered proteins. Under the metabolism and signalling section Reactome, ConsensusPathDB, HMDB and CAZy are major returning resources. In microbial and viral genomes taxonomy and systematics are well covered by LPSN, TYGS and GTDB. Genomics resources include Ensembl, Ensembl Genomes and UCSC Genome Browser. Major returning pharmacology resource names include the IUPHAR/BPS guide and the Therapeutic Target Database. New plant databases include PlantGSAD for gene lists and qPTMplants for post-translational modifications. The entire Database Issue is freely available online on the Nucleic Acids Research website (https://academic.oup.com/nar). Our latest update to the NAR online Molecular Biology Database Collection brings the total number of entries to 1645. Following last year's major cleanup, we have updated 317 entries, listing 89 new resources and trimming 80 discontinued URLs. The current release is available at http://www.oxfordjournals.org/nar/database/c/., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA associates with RNA-binding proteins (RBPs) from synthesis to decay, forming dynamic ribonucleoproteins (RNPs). In spite of the preeminent role of RBPs regulating RNA fate, the scope of cellular RBPs has remained largely unknown. We have recently developed a novel and comprehensive method to identify the repertoire of active RBPs of cultured cells, called RNA interactome capture. Using in vivo UV cross-linking on cultured cells, proteins are covalently bound to RNA if the contact between the two is direct ("zero distance"). Protein-RNA complexes are purified by poly(A) tail-dependent oligo(dT) capture and analyzed by quantitative mass spectrometry. Because UV irradiation is applied to living cells and purification is performed using highly stringent washes, RNA interactome capture identifies physiologic and direct protein-RNA interactions. Applied to HeLa cells, this protocol revealed the near-complete repertoire of RBPs, including hundreds of novel RNA binders. Apart from its RBP discovery capacity, quantitative and comparative RNA interactome capture can also be used to study the responses of the RBP repertoire to different physiological cues and processes, including metabolic stress, differentiation, development, or the response to drugs.

Proteome-wide discovery of in vivo metacaspase substrates can be obtained by positional proteomics approaches such as N-terminal COFRADIC, for example by comparing the N-terminal proteomes (or N-terminomes) of wild-type plants to transgenic plants not expressing a given metacaspase. In this chapter we describe a protocol for the preparation of plant tissue proteomes, including differential isotopic labelling allowing for a comparison of in vivo N-terminomes that serves as the starting point for N-terminal COFRADIC studies.

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed.

Mitochondria are dynamic organelles with activities that adjust to altering physiological conditions and variable metabolic demands. A conserved proteolytic system present within the organelle exerts essential functions during the biogenesis of mitochondria and ensures the maintenance of organellar activities under varying conditions. Proteases dependent on adenosine triphosphate, in concert with oligopeptidases, degrade nonassembled or damaged proteins in various subcompartments of mitochondria, preventing their accumulation and possibly deleterious effects on mitochondrial functions. Although an increasing number of mitochondrial peptidases are characterized and functionally linked to diverse cellular processes, only limited information is available on the stability of the mitochondrial proteome and the turnover rates of individual proteins. We describe experimental approaches in the yeast Saccharomyces cerevisiae and in mice, allowing analysis of the proteolytic breakdown of mitochondrial proteins individually or on a proteomewide scale.

Rapid advances have been made in recent years in understanding the genetic makeup of mankind. The human genome project has identified approximately 32,000 genes that occur in humans. It is now possible to perform genetic profiling with many of the genes to understand embryogenesis, growth and development, the normal state, senescence, diseases, and tumorogenesis. Techniques in molecular diagnostics are becoming available that will expand the ability to provide more precise diagnoses, predict response to treatment, evaluate treatment, and predict prognosis and outcome. Genetic profiling will, in the future, direct therapy by providing specific targets for development of medications, antibodies, and gene therapy. More recently, the attention of the scientific community has turned toward the gene products within the cell and tissue matrix, namely proteins. The field of proteomics is an evolving area, which may shed light on the proteins associated with diseases and tumors. This will again provide a mechanism for creating personalized, designer therapies for individual patients or groups of patients with similar diseases based on expression profiling. The final avenue of exploration in understanding cell function is the metabolites that occur as the end products of cellular function (metabolome). These metabolites, or improper degradation of cellular proteins, may lead to disease and neoplasia. The role of the pathologist in expression profiling for diseases and tumorigenesis is of considerable importance in providing diagnostic information and predicting outcome based on pathobiologic features of diseases and tumors.

The human genome project and the technological breakthroughs it has produced have moved the field of molecular medicine forward with breathtaking speed. This will impact not only the advance of scientific discoveries and the way science is conducted but also the clinical practice of medicine. In this review we explain the basic principles of these new technologies. Their potential use and impact are demonstrated by using diabetes mellitus as an example of a common and serious medical disorder. Finally, several potentially adverse consequences of "excessive" knowledge are discussed.

Objectives: The increasing production of molecular biology data in the post-genomic era, and the proliferation of databases that store it, require the development of an integrative layer in database services to facilitate the synthesis of related information. The solution of this problem is made more difficult by the absence of universal identifiers for biological entities, and the breadth and variety of available data., Methods: Integr8 was modelled using UML (Universal Modelling Language). Integr8 is being implemented as an n-tier system using a modern object-oriented programming language (Java). An object-relational mapping tool, OJB, is being used to specify the interface between the upper layers and an underlying relational database., Results: The European Bioinformatics Institute is launching the Integr8 project. Integr8 will be an automatically populated database in which we will maintain stable identifiers for biological entities, describe their relationships with each other (in accordance with the central dogma of biology), and store equivalences between identified entities in the source databases. Only core data will be stored in Integr8, with web links to the source databases providing further information., Conclusions: Integr8 will provide the integrative layer of the next generation of bioinformatics services from the EBI. Web-based interfaces will be developed to offer gene-centric views of the integrated data, presenting (where known) the links between genome, proteome and phenotype.

The emergence of proteomics, the large-scale analysis of proteins, has been inspired by the realization that the final product of a gene is inherently more complex and closer to function than the gene itself. Shortfalls in the ability of bioinformatics to predict both the existence and function of genes have also illustrated the need for protein analysis. Moreover, only through the study of proteins can posttranslational modifications be determined, which can profoundly affect protein function. Proteomics has been enabled by the accumulation of both DNA and protein sequence databases, improvements in mass spectrometry, and the development of computer algorithms for database searching. In this review, we describe why proteomics is important, how it is conducted, and how it can be applied to complement other existing technologies. We conclude that currently, the most practical application of proteomics is the analysis of target proteins as opposed to entire proteomes. This type of proteomics, referred to as functional proteomics, is always driven by a specific biological question. In this way, protein identification and characterization has a meaningful outcome. We discuss some of the advantages of a functional proteomics approach and provide examples of how different methodologies can be utilized to address a wide variety of biological problems.

Clinical researchers, practicing physicians, patients, and the general public now live in a world in which the 2.9 billion nucleotide codes of the human genome are available as a resource for scientific discovery. Some of the findings from the sequencing of the human genome were expected, confirming knowledge presaged by many decades of research in both human and comparative genetics. Other findings are unexpected in their scientific and philosophical implications. In either case, the availability of the human genome is likely to have significant implications, first for clinical research and then for the practice of medicine. This article provides our reflections on what the new genomic knowledge might mean for the future of medicine and how the new knowledge relates to what we knew in the era before the availability of the genome sequence. In addition, practicing physicians in many communities are traditionally also ambassadors of science, called on to translate arcane data or the complex ramifications of biology into a language understood by the public at large. This article also may be useful for physicians who serve in this capacity in their communities. We address the following issues: the number of protein-coding genes in the human genome and certain classes of noncoding repeat elements in the genome; features of genome evolution, including large-scale duplications; an overview of the predicted protein set to highlight prominent differences between the human genome and other sequenced eukaryotic genomes; and DNA variation in the human genome. In addition, we show how this information lays the foundations for ongoing and future endeavors that will revolutionize biomedical research and our understanding of human health.

The identification of coding sequences in a number of species, including human in the near future, has ushered in the post-genome era. In this era, technologies are becoming available that allow the profiling of tissues and cell populations at the genomic, transcriptomic and proteomic levels. The molecular analysis of tissues at all three levels has been referred to as operomics. This review covers some basic technologies for operomics and their application to some lymphoid disorders. It is proposed that no one type of analysis is fully informative and that information that can be derived from the different compartments encompassed in operomics is complementary. Prospects for introducing such profiling technologies into the clinical laboratory will depend on their robustness, their user friendliness and the clinical relevance of the added information they provide, which cannot be captured through other technologies in use in the clinical laboratory.

Recent developments in mass spectrometry (MS) provide scientists with an established analytical tool that addresses the demands for rapid, accurate and cost effective analyses of biomolecules. These advances clearly accelerate the rate and success of protein identification, genetic sequencing, determining biological variances and drug discovery. This review is intended to illustrate how matrix-assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) MS is typically used to generate information about biomolecules under investigation. Additionally, examples will be used to describe the steps involved in preparing samples for MALDI TOF and obtaining answers through data management.

The goal of molecular ophthalmology is the early detection and therapeutic treatment of eye disease. Genomic technologies have profoundly enhanced the discovery of ocular disease candidate genes. Proteomics, the protein cognate of genomic technology, offers a means to monitor changes in the expression of a given ocular protein(s) and its post-translational modification, identify novel therapeutic targets and evaluate pharmacological effects on a given metabolic pathway. Using both tissue and cultured cells, numerous laboratories have begun to catalogue changes in ocular protein expression in normal, diseased and ageing subjects. Herein, we review published proteomic literature in the broad context of ophthalmic diseases involving various tissues of the eye.

Background Patients with intrahepatic cholangiocarcinoma (ICC) are prone to recurrence and poor survival. Targeted therapy related to isocitrate dehydrogenase (IDH) is an extremely important treatment. IDH1 and IDH2 mutations are generally thought to have similar effects on the tumor landscape. However, it is doubtful whether these 2 mutations have exactly the same effects on tumor cells and the tumor microenvironment. Methods All collected tumor samples were subjected to simultaneous whole-exon sequencing and proteome sequencing. Results IDH1 mutations accounted for 12.2%, and IDH2 mutations accounted for 5.5%, all missense mutations. Tumors with IDH mutations had lower proportions of KRAS and TP53 mutations. Mutated genes were obviously enriched in the kinase pathway in the tumors with IDH2 mutations. The signaling pathways were mainly enriched in the activation of cellular metabolic activities and an increase of inhibitory immune cells in the tumors with IDH mutations. Moreover, tumors had unique enrichment in DNA repair in IDH1 mutants and secretion of biological molecules in IDH2 mutants. Inhibitory immune cells might be more prominent in IDH2 mutants, and the expression of immune checkpoints PVR and HLA-DQB1 was more prominent in IDH1 mutants. IDH mutants were more related to metabolism-related and inflammation-immune response clusters, and some belonged to the DNA replication and repair cluster. Conclusions These results revealed the differential IDH1 and IDH2 mutation-related landscapes, and we have provided an important reference database to guide ICC treatment. [ABSTRACT FROM AUTHOR]

Background: Despite the significant progress in the treatment of Acute Lymphoblastic Leukemia (ALL) in children, it still remains as one of the most challenging malignancies in adults. Identification of new biomarkers may improve the management of adult ALL. Proteins expressed on the cell surface can be considered as disease-associated biomarkers with potential for diagnosis and targeted therapies. Thus, membrane proteome studies give essential information about the disease-related biomarkers. Methods: We applied 2-dimensional blue-native SDS-PAGE technique followed by MALDI-TOF/TOF-mass spectrometry to study the cell membrane proteome of peripheral blood mononuclear cells of adult B-ALL patients in comparison to that of the healthy controls. Results: Sixty seven differentially expressed protein spots were detected, among them 52 proteins were found to be up-regulated but the other 15 proteins were downregulated in B-ALL. Five differentially expressed proteins, involved in energy metabolism pathways, were detected in B-ALL patients compared to the healthy control group. Conclusion: Differentially expressed proteins provide an insight into the molecular biology of B-ALL. Further studies must be done to confirm our data to be considered as potential targets for detection and treatment of B-ALL. [ABSTRACT FROM AUTHOR]

Proteomics is one of the applied sciences in biology, this century. Using the mass spectrometer device and the development of databases and bioinformatics tools, a fundamental evolution has taken place in molecular biology and new perspectives have been found in agriculture, pharmaceuticals, molecular biology, etc. Two-dimensional gel electrophoresis methods are a popular technique for protein separation because they allow researchers to quantify protein changes on a large scale. Proteomics is a large-scale evaluation of proteins. The term proteomics was produced in 1997 after the introduction of genomics. Although the use of databases and bioinformatics to interpret the results of proteomic findings is being seriously developed. In the study of huge proteomic data, it is very desirable to use the capacity of multivariate statistical methods, due to having many and extensive variables, because these multivariate statistical methods provide the possibility of statistical analysis of several variables, therefore, the use of this statistical technique recently, it has attracted the attention of proteomics scientists. In addition to understanding the tools of proteomics, we are looking for the practical use of proteomics, especially in agriculture, because today the pressure is on plant breeders to provide "smart plant varieties" that are more suitable genotypes with the ability to tolerate biotic and abiotic stress a wider range of climate changes for dealing with the food insecurity of future generations. Therefore, modern plant breeders need precise genetic modification with a gene tracking system for the modified trait. The only caveat in the application of proteomics in biotechnology applications is that genetic modification must be expressed at the protein level. The purpose of this research is to examine the concepts of some main topics in the field of proteomics in simple language, and of course, case studies of proteomics in plants, especially in industrial oilseed plants. [ABSTRACT FROM AUTHOR]

The oral epithelium's normal morphological structure and function play an important role in maintaining oral homeostasis, among which microbiota and chronic stress are key contributing factors. However, the effects of microbiota and chronic stress on the morphological structures and molecular function of oral homeostasis remain unclear. In this study, morphological staining was used to compare the tongue structure of specific pathogen-free and germfree mice, and an integrated multi-omics analysis based on transcriptomics, proteomics, and metabolomics was performed to investigate the regulatory mechanisms of microbiota and chronic stress on oral homeostasis. We found that the morphological structure of the tongue in germ-free mice was disordered compared with in specific pathogen-free mice, especially in the epithelium. Multi-omics analysis indicated that differentially expressed molecules of the tongue between germ-free and specific pathogen-free mice were significantly enriched in the mitochondrial metabolic process and immune response. Interestingly, microbiota also significantly influenced the permeability of the oral epithelial barrier, represented by the differential expression of keratinization, and cell adhesion molecules. It was worth noting that the above changes in the tongue between specific pathogen-free and germ-free mice were more significant after chronic stress. Collectively, this is the first study to reveal that the microbiota might maintain oral homeostasis by reshaping the structure of the oral epithelial barrier and changing the function of molecular biology, a process that may be driven by the immune response and mitochondrial metabolic process of oral tissue. Furthermore, chronic stress can enhance the regulatory effects of microbiota on oral homeostasis. [ABSTRACT FROM AUTHOR]

Secreted amyloid precursor protein alpha (sAPPα) processed from a parent human brain protein, APP, can modulate learning and memory. It has potential for development as a therapy preventing, delaying, or even reversing Alzheimer's disease. In this study a comprehensive analysis to understand how it affects the transcriptome and proteome of the human neuron was undertaken. Human inducible pluripotent stem cell (iPSC)-derived glutamatergic neurons in culture were exposed to 1 nM sAPPα over a time course and changes in the transcriptome and proteome were identified with RNA sequencing and Sequential Window Acquisition of All THeoretical Fragment Ion Spectra-Mass Spectrometry (SWATH-MS), respectively. A large subset (∼30%) of differentially expressed transcripts and proteins were functionally involved with the molecular biology of learning and memory, consistent with reported links of sAPPα to memory enhancement, as well as neurogenic, neurotrophic, and neuroprotective phenotypes in previous studies. Differentially regulated proteins included those encoded in previously identified Alzheimer's risk genes, APP processing related proteins, proteins involved in synaptogenesis, neurotransmitters, receptors, synaptic vesicle proteins, cytoskeletal proteins, proteins involved in protein and organelle trafficking, and proteins important for cell signalling, transcriptional splicing, and functions of the proteasome and lysosome. We have identified a complex set of genes affected by sAPPα, which may aid further investigation into the mechanism of how this neuroprotective protein affects memory formation and how it might be used as an Alzheimer's disease therapy. [ABSTRACT FROM AUTHOR]

Proteins are central to all of the processes of life. For their activity, they almost invariably need to interact with other macromolecules, be they nucleic acids, membranes, glycans, or other proteins. The interaction between proteins is indeed the most common mode of macromolecular interaction underpinning living systems. To understand these systems at a molecular level, it is therefore essential to identify and characterize their constituent protein-protein interactions. Despite an unprecedented growth in our knowledge of complete proteomes across all domains of life, both at the sequence level and increasingly at the structure level, the inherently low accuracy and molecular resolution of many techniques have made the characterization of protein-protein interactions one of the grand challenges of molecular biology. In this review, we survey both computational and experimental techniques for the medium- to high-throughput characterization of protein-protein interactions and discuss the potential of integrative approaches, given recent advances in sequence analysis and structure prediction. [Display omitted] Proteins are central agents of life, and for their activity, they need interaction with other proteins. Describing these interactions is thus essential for a molecular understanding of living processes. Here, Elhabashy et al. review the current methods for the characterization of protein-protein interactions and discuss them in light of recent advances. [ABSTRACT FROM AUTHOR]

Objective: Odontogenic keratocyst (OKC) is a benign lesion that tends to recur after surgical treatment. In an attempt to clarify the molecular basis underlining the OKC pathobiology, we aimed to analyze its proteomic profile. Materials and Methods: We compared the proteomic profiles of five OKC and matched normal oral mucosa by using liquid chromatography–tandem mass spectrometry (LC‐MS/MS). Then, we performed enrichment analysis and a literature search for the immunoexpression of the proteomics targets. Results: We identified 1,150 proteins and 72 differently expressed proteins (log2 fold change ≥ 1.5; p <.05). Twenty‐seven peptides were exclusively detected in the OKC samples. We found 35 enriched pathways related to cell differentiation and tissue architecture, including keratinocyte differentiation, keratinization, desmosome, and extracellular matrix (ECM) organization and degradation. The immunoexpression information of 11 out of 50 proteins identified in the enriched pathways was obtained. We found the downregulation of four desmosomal proteins (JUP, PKP1, PKP3, and PPL) and upregulation of ECM proteases (MMP‐2, MMP‐9, and cathepsins). Conclusions: Proteomic analysis strengthened the notion that OKC cells have a similar proteomic profile to oral keratinocytes. Contextual investigation of the differentially expressed proteins revealed the deregulation of desmosome proteins and ECM degradation as important alterations in OKC pathobiology. [ABSTRACT FROM AUTHOR]