Proteomics of osteosarcoma

Osteosarcoma (OS) is the most common primary malignant tumor of bone and the third most common cancer in childhood and adolescence. Nowadays, early diagnosis, drug resistance and recurrence of the disease represent the major challenges in OS treatment. Post-genomics, and in particular proteomic technologies, offer an invaluable opportunity to address the level of biological complexity expressed by OS. Although the main goal of OS oncoproteomics is focused on diagnostic and prognostic biomarker discovery, in this review we describe and discuss global protein profiling approaches to other aspects of OS biology and pathophysiology, or to investigate the mechanism of action of chemotherapeutics. In addition, we present proteomic analyses carried out on OS cell lines as in vitro models for studying osteoblastic cell biology and the attractive opportunity offered by proteomics of OS cancer stem cells.     

Brain asymmetry: both sides of the story

Biological systems demonstrate asymmetry, while lateralization has been observed from humans to lower animals structurally, functionally and behaviorally. This may be derived from evolutionary, genetic, developmental, epigenetic and pathologic factors. However, brain structure and function is complex, and macroscopic or microscopic asymmetries are hard to discern from random fluctuations. In this article, we discuss brain laterality and lateralization, beginning with a brief review of the literature on brain structural and functional asymmetries. We conclude with methods to detect and quantify asymmetry, focusing on neuroproteomics, for retrieval of protein-expression patterns, as a method of diagnosis and treatment monitoring. We suggest inter-hemispheric differential proteomics as a valid method to assess the experimental and biological variations in the healthy brain, and neurologic and neuropsychiatric disorders.     

Proteomic analysis of phosphorylation in cancer

Constitutive activity of kinases is known to be crucial for a tumor to maintain its malignant phenotype, a phenomenon which is often referred to as oncogene addiction. The in-depth analysis of aberrant signaling pathways by the analysis of protein phosphorylation has become feasible through recent advances in proteomics technology. In this article we will review developments in the field of phosphoproteomics and its application in cancer research. The most widely used technologies for the generic enrichment of phosphopeptides are discussed as well as targeted approaches for the analysis of a specific subset of phosphopeptides. Validation experiments of phosphorylation sites using targeted mass spectrometry are also explained. Finally, we will highlight applications of phosphoproteomic technology in cancer research using cell lines and tissue.     

Isotopically labeled proteome as an internal standard for multiple reaction monitoring-based biomarker quantification

Multiple reaction monitoring is a mass spectrometry technology used to selectively identify and quantify a known molecule in a complex mixture. The technology has gained favor in proteomic applications, especially for biomarker quantification in human samples. For this purpose, employment of internal standard consisting of isotopically (heavy) labeled proteins is currently considered the best way of normalizing sample preparation and correcting for different ionization efficiencies. However, synthesis of heavy-labeled proteins is considered laborious and expensive. The work outlined here presents an efficient strategy of utilizing isotope-labeled amino acids in cell culture to produce heavy-labeled proteins. These are then spiked into serum and serve as internal standards to relatively quantify a large number of target proteins. The method has been applied to quantify 72 proteins in the sera of pancreatic cancer patients with remarkable efficiency and accuracy.     

Potential blood biomarkers for stroke

Stroke is one of the most common causes of death worldwide and a major cause of acquired disability in adults. Despite advances in research during the last decade, prevention and treatment strategies still suffer from significant limitations, and therefore new theoretical and technical approaches are required. Technological advances in the proteomic and metabolomic areas, during recent years, have permitted a more effective search for novel biomarkers and therapeutic targets that may allow for effective risk stratification and early diagnosis with subsequent rapid treatment. This review provides a comprehensive overview of the latest candidate proteins and metabolites proposed as new potential biomarkers in stroke.     

Proteomic analysis in allergy and intolerance to wheat products

Owing to its extensive use in the human diet, wheat is among the most common causes of food-related allergies and intolerances. Allergies to wheat are provoked by ingestion, inhalation or contact with either the soluble or the insoluble gluten proteins in wheat. Gluten proteins, and particularly the gliadin fraction, are also the main factor triggering celiac disease, a common enteropathy induced by ingestion of wheat gluten proteins and related prolamins from oat, rye and barley in genetically susceptible individuals. The role of gliadin and of its derived peptides in eliciting the adverse reactions in celiac disease are still far from being completely explained. Owing to its unique pathogenesis, celiac disease is widely investigated as a model immunogenetic disorder. The structural characterization of the injuring agents, the gluten proteins, assumes a particular significance in order to deepen the understanding of the events that trigger this and similar diseases at the molecular level. Recent developments in proteomics have provided an important contribution to the understanding of several basic aspects of wheat protein-related diseases. These include: the identification of gluten fractions and derived peptides involved in wheat allergy and intolerance, including celiac disease, and the elucidation of their mechanism of toxicity; the development and validation of sensitive and specific methods for detecting trace amounts of gluten proteins in gluten-free foods for intolerant patients; and the formulation of completely new substitute foods and ingredients to replace the gluten-based ones. In this article, the main aspects of current and prospective applications of mass spectrometry and proteomic technologies to the structural characterization of gluten proteins and derived peptides are critically presented, with a focus on issues related to their detection, identification and quantification, which are relevant to the biochemical, immunological and toxicological aspects of wheat intolerance.     

Proteomic analysis in the identification of allergenic molecules

Conventional and innovative strategies can be exploited to identify and characterize new allergenic proteins. With the aim of obtaining suggestions for future improvements, this article describes our attempt to understand and describe some of the advantages and pitfalls of the methodologies and procedures often used in this field. The analysis includes the protein extract preparation, starting from the allergenic source, the separation of the proteins contained in a mixture and the detection, identification and characterization of IgE-binding molecules. Classic and emerging proteomic technologies, including mass spectrometry-based methodologies, Edman degradation procedure, microarray-based techniques and bioinformatics search strategies, have been explored. A comparative analysis of biochemistry-based proteomics and molecular biology strategies has also been given.     

A combined proteomics and computational approach provides a better understanding of HCV-induced liver disease

Evaluation of: Diamond DL, Krasnoselsky AL, Burnum KE et al. Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation. Hepatology 56(1), 28–38 (2012).

HCV is a major cause of chronic liver disease worldwide and is a formidable therapeutic challenge. Recently, Diamond et al. analyzed the proteomic profiles of liver samples from HCV-positive liver transplant recipients, supplemented with an independent metabolite analysis. They used a computational approach, which highlighted the enriched functional themes and topological attributes associated with the protein association network based on their clinical data and suggested a crucial role of oxidative stress in fibrosis progression in HCV infection. Their findings provide new insights into the mechanisms that regulate the progression of HCV-associated liver fibrosis, which may be useful for identification of suitable biomarkers to evaluate the onset and severity of hepatic fibrosis and the development of new therapeutic and anti-HCV strategies.     

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.     

Prodromo della Flora Umbra

Defense responses of plants are activated not only in the wounded tissues but also in the remote parts of the plants. Two different methyl jasmonate (MeJA) treatments were conducted, i.e., MeJA solution spraying of entire rosettes leaves and pasting leaf surface with lanolin squares containing MeJA. Glucosinolate profiles in leaves were similar using the two methods of MeJA treatment except for indole glucosinolates. The glucosinolate profiles in local and systemic leaves showed that the accumulation of glucosinolates in systemic leaves were delayed comparing with those in local treated leaves. Comparative proteomics were used to investigate the molecular processes underlying the glucosinolate changes in response to local and systemic MeJA induction. A total of 83 unique proteins were detected as differentially expressed between the local and systemic leaves. Functional analysis showed that redirection of metabolism from growth to defense was differentially regulated in local and systemic MeJA induction. The higher contents of indole glucosinolates in systemic leaves might arise from the induction of a long-distance signal produced in local leaves as well as from JA synthesized in systemic leaves.