Proteomes — Open Access Proteomics Journal

Legumes are a large and economically important family, containing a variety of crop plants. Alongside different cereals, some fruits, and tropical roots, a number of leguminosae evolved for millennia as crops with human society. One of these legumes is Pisum sativum L., the common garden pea. In the past, breeding has been largely selective on improved above-ground organs. However, parameters, such as root-growth, which determines acquisition of nutrients and water, have largely been underestimated. Although the genome of P. sativum is still not fully sequenced, multiple proteomic studies have been published on a variety of physiological aspects in the last years. The presented work focused on the connection between root length and the influence of the microsomal root proteome of four different pea cultivars after five days of germination (cultivar Vroege, Girl from the Rhineland, Kelvedon Wonder, and Blauwschokker). In total, 60 proteins were identified to have significantly differential abundances in the four cultivars. Root growth of five-days old seedlings and their microsomal proteome revealed a similar separation pattern, suggesting that cultivar-specific root growth performance is explained by differential membrane and ribosomal protein levels. Hence, we reveal and discuss several putative root growth protein markers possibly playing a key role for improved primary root growth breeding strategies. Full article

Since the publication of one of the first studies using 2D gel electrophoresis by Patrick H. O’Farrell in 1975, several other studies have used that method to evaluate cellular responses to different physicochemical variations. In environmental microbiology, bacterial adaptation to cold environments is a “hot topic” because of its application in biotechnological processes. As in other fields, gel-based and gel-free proteomic methods have been used to determine the molecular mechanisms of adaptation to cold of several psychrotrophic and psychrophilic bacterial species. In this review, we aim to describe and discuss these main molecular mechanisms of cold adaptation, referencing proteomic studies that have made significant contributions to our current knowledge in the area. Furthermore, we use Exiguobacterium antarcticum B7 as a model organism to present the importance of integrating genomic, transcriptomic, and proteomic data. This species has been isolated in Antarctica and previously studied at all three omic levels. The integration of these data permitted more robust conclusions about the mechanisms of bacterial adaptation to cold. Full article

The tomato (Solanum lycopersicum) ripening process from mature green (MG) to turning and then to red stages is accompanied by the occurrences of physiological and biochemical reactions, which ultimately result in the formation of the flavor, color and texture of ripe fruits. The two trivalent metal ions Al³⁺ and La³⁺ are known to induce different levels of phytotoxicity in suppressing root growth. This paper aims to understand the impacts of these two metal ions on tomato fruit proteomes. Tomato ‘Micro-Tom’ plants were grown in a hydroponic culture system supplemented with 50 μM aluminum sulfate (Al₂ (SO4)₃.18H₂O) for Al³⁺ or La₂(SO₄)₃ for La³⁺. Quantitative proteomics analysis, using isobaric tags for relative and absolute quantitation, were performed for fruits at MG, turning and red stages. Results show that in MG tomatoes, proteins involved in protein biosynthesis, photosynthesis and primary carbohydrate metabolisms were at a significantly lower level in Al-treated compared to La-treated plants. For the turning and red tomatoes, only a few proteins of significant differences between the two metal treatments were identified. Results from this study indicate that compared to La³⁺, Al³⁺ had a greater influence on the basic biological activities in green tomatoes, but such an impact became indistinguishable as tomatoes matured into the late ripening stages. Full article

De novo sequencing of tandem (MS/MS) mass spectra represents the only way to determine the sequence of proteins from organisms with unknown genomes, or the ones not directly inscribed in a genome—such as antibodies, or novel splice variants. Top-down mass spectrometry provides new opportunities for analyzing such proteins; however, retrieving a complete protein sequence from top-down MS/MS spectra still remains a distant goal. In this paper, we review the state-of-the-art on this subject, and enhance our previously developed Twister algorithm for de novo sequencing of peptides from top-down MS/MS spectra to derive longer sequence fragments of a target protein. Full article

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Patient survival has remained largely the same for the past 20 years, with therapies causing significant health, cognitive, behavioral and developmental complications for those who survive the tumor. In this study, we profiled the total transcriptome and proteome of two established MB cell lines, Daoy and UW228, using high-throughput RNA sequencing (RNA-Seq) and label-free nano-LC-MS/MS-based quantitative proteomics, coupled with advanced pathway analysis. While Daoy has been suggested to belong to the sonic hedgehog (SHH) subtype, the exact UW228 subtype is not yet clearly established. Thus, a goal of this study was to identify protein markers and pathways that would help elucidate their subtype classification. A number of differentially expressed genes and proteins, including a number of adhesion, cytoskeletal and signaling molecules, were observed between the two cell lines. While several cancer-associated genes/proteins exhibited similar expression across the two cell lines, upregulation of a number of signature proteins and enrichment of key components of SHH and WNT signaling pathways were uniquely observed in Daoy and UW228, respectively. The novel information on differentially expressed genes/proteins and enriched pathways provide insights into the biology of MB, which could help elucidate their subtype classification. Full article

The separation of ampholytic components according to isoelectric point has played an important role in isolating, reducing complexity and improving peptide and protein detection. This brief review outlines the basics of isoelectric focusing, including a summary of the historical achievements and considerations in experimental design. Derivative methodologies of isoelectric focusing are also discussed including common detection methods used. Applications in a variety of fields using isoelectric point based separations are provided as well as an outlook on the field for future studies. Full article

Post-translational modifications (PTMs) play a key role in regulating protein function, yet their identification is technically demanding. Here, we present a straightforward workflow to systematically identify post-translationally modified proteins based on two-dimensional gel electrophoresis. Upon colloidal Coomassie staining the proteins are partially transferred, and the investigated PTMs are immunodetected. This strategy allows tracking back the immunopositive antigens to the corresponding spots on the original gel, from which they are excised and mass spectrometrically identified. Candidate proteins are validated on the same membrane by immunodetection using a second fluorescence channel. We exemplify the power of partial immunoblotting with the identification of lysine-acetylated proteins in myelin, the oligodendroglial membrane that insulates neuronal axons. The excellent consistency of the detected fluorescence signals at all levels allows the differential comparison of PTMs across multiple conditions. Beyond PTM screening, our multi-level workflow can be readily adapted to clinical applications such as identifying auto-immune antigens or host-pathogen interactions. Full article

The ability to selectively amplify the detection signals for targets over interferences is crucial when analyzing proteins in a complicated sample matrix. Here, we describe a targeted enlargement strategy that can amplify the light-scattering signal from aptamer-functionalized gold nanoparticles (Apt-AuNP) with high specificity for quantitative protein analysis. This strategy is achieved by labeling target proteins with competitively protected Apt-AuNP probes and enlarging the probes with gold enhancement. This competitive protection strategy could effectively eliminate nonspecific protein adsorptions from a sample matrix, leading to a highly specific labeling of the target protein. As a result, the subsequent amplification of the light-scattering signal by gold enhancement only occurs in the presence of the target protein. This strategy was successfully demonstrated by analyzing human α-thrombin in human serum samples in a Western blot format. Full article

Proteomics is a field of growing importance in animal and aquatic sciences. Similar to other proteomic approaches, top-down proteomics is slowly making its way within the vast array of proteomic approaches that researchers have access to. This opinion and mini-review article is dedicated to top-down proteomics and how its use can be of importance to animal and aquatic sciences. Herein, we include an overview of the principles of top-down proteomics and how it differs regarding other more commonly used proteomic methods, especially bottom-up proteomics. In addition, we provide relevant sections on how the approach was or can be used as a research tool and conclude with our opinions of future use in animal and aquatic sciences. Full article

The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs) can regulate protein activity and localization as well as protein–protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future. Full article

Resurrection plants can tolerate extreme water loss. Substantial sugar accumulation is a phenomenon in resurrection plants during dehydration. Sugars have been identified as one important factor contributing to desiccation tolerance. Phylogenetic diversity of resurrection plants reflects the diversity of sugar metabolism in response to dehydration. Sugars, which accumulate during dehydration, have been shown to protect macromolecules and membranes and to scavenge reactive oxygen species. This review focuses on the performance of enzymes participating in sugar metabolism during dehydration stress. The relation between sugar metabolism and other biochemical activities is discussed and open questions as well as potential experimental approaches are proposed. Full article

Oral cancer refers to malignancies that have higher morbidity and mortality rates due to the late stage diagnosis and no early detection of a reliable diagnostic marker, while oral squamous cell carcinoma (OSCC) is amongst the world’s top ten most common cancers. Diagnosis of cancer requires highly sensitive and specific diagnostic tools which can support untraceable hidden sites of OSCC, yet to be unleashed, for which plenty of biomarkers are identified; the most recommended biomarker detection medium for OSCC includes biological fluids, such as blood and saliva. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being a non-invasive technique to follow, by analysing the malignant cells’ molecular pathology obtained from saliva through proteomic, genomic and transcriptomic approaches. However, protein biomarkers provide an immense potential for developing novel marker-based assays for oral cancer, hence this current review offers an overall focus on the discovery of a panel of candidates as salivary protein biomarkers, as well as the proteomic tools used for their identification and their significance in early oral cancer detection. Full article

Phosphorylation plays a critical role in regulating protein function and thus influences a vast spectrum of cellular processes. With the advent of modern bioanalytical technologies, examination of protein phosphorylation on a global scale has become one of the major research areas. Phosphoproteins are found in biological fluids and interrogation of the phosphoproteome in biological fluids presents an exciting opportunity for discoveries that hold great potential for novel mechanistic insights into protein function in health and disease, and for translation to improved diagnostic and therapeutic approaches for the clinical setting. This review focuses on phosphoproteome discovery in selected human biological fluids: serum/plasma, urine, cerebrospinal fluid, saliva, and bronchoalveolar lavage fluid. Bioanalytical workflows pertinent to phosphoproteomics of biological fluids are discussed with emphasis on mass spectrometry-based approaches, and summaries of studies on phosphoproteome discovery in major fluids are presented. Full article

The invention of the microscope has been fundamental for the understanding of tissue architecture and subcellular structures. With the advancement of higher magnification microscopes came the development of various molecular biology tools such as Förster resonance energy transfer (FRET) and in situ proximity ligation assay (in situ PLA) to monitor protein interactions. Microscopy has become a commonly used method for the investigation of molecular events within the cell, for the identification of key players in signaling networks, and the activation of these pathways. Multiple approaches are available for functional analyses in single cells. They provide information not only on the localization of proteins at a given time point, but also on their expression levels and activity states, allowing us to pinpoint hallmarks of different cellular identities within tissues in health and disease. Clever solutions to increase the sensitivity of molecular tools, the possibilities for multiplexing, as well as image resolution have recently been introduced; however, these methods have their pros and cons. Therefore, one needs to carefully consider the biological question of interest along with the nature of the sample before choosing the most suitable method or combination of methods. Herein, we review a few of the most exciting microscopy-based molecular techniques for proteomic analysis and cover the benefits as well as the disadvantages of their use. Full article

During the past decade, the hand-in-hand development of biotechnology and bioinformatics has enabled a view of the function of the red blood cell that surpasses the supply of oxygen and removal of carbon dioxide. Comparative proteomic inventories have yielded new clues to the processes that regulate membrane–cytoskeleton interactions in health and disease, and to the ways by which red blood cells communicate with their environment. In addition, proteomic data have revealed the possibility that many, hitherto unsuspected, metabolic processes are active in the red blood cell cytoplasm. Recent metabolomic studies have confirmed and expanded this notion. Taken together, the presently available data point towards the red blood cell membrane as the hub at which all regulatory processes come together. Thus, alterations in the association of regulatory proteins with the cell membrane may be a sine qua non for the functional relevance of any postulated molecular mechanism. From this perspective, comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red blood cell homeostasis. Additionally, this perspective provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood. Full article

The development of a range of sub-proteomic approaches to the plant cell wall has identified many of the cell wall proteins. However, it remains difficult to elucidate the precise biological role of each protein and the cell wall dynamics driven by their actions. The plant protoplast provides an excellent means not only for characterizing cell wall proteins, but also for visualizing the dynamics of cell wall regeneration, during which cell wall proteins are secreted. It therefore offers a unique opportunity to investigate the de novo construction process of the cell wall. This review deals with sub-proteomic approaches to the plant cell wall through the use of protoplasts, a methodology that will provide the basis for further exploration of cell wall proteins and cell wall dynamics. Full article

Here, approaches that allow disclosure of the information hidden inside and outside of two-dimensional gel electrophoresis (2DE) are described. Experimental identification methods, such as mass spectrometry of high resolution and sensitivity (MALDI-TOF MS and ESI LC-MS/MS) and immunodetection (Western and Far-Western) in combination with bioinformatics (collection of all information about proteoforms), move 2DE to the next level of power. The integration of these technologies will promote 2DE as a powerful methodology of proteomics technology. Full article

Mass spectrometry imaging (MSI) is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i) allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii) was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation. Full article

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine. Full article