Search

Tumor heterogeneity refers to the diversity observed among tumor cells: both between different tumors (inter-tumor heterogeneity) and within a single tumor (intra-tumor heterogeneity). These cells can display distinct morphological and phenotypic characteristics, including variations in cellular morphology, metastatic potential and variability treatment responses among patients. Therefore, a comprehensive understanding of such heterogeneity is necessary for deciphering tumor-specific mechanisms that may be diagnostically and therapeutically valuable. Innovative and multidisciplinary approaches are needed to understand this complex feature. In this context, proteogenomics has been emerging as a significant resource for integrating omics fields such as genomics and proteomics. By combining data obtained from both Next-Generation Sequencing (NGS) technologies and mass spectrometry (MS) analyses, proteogenomics aims to provide a comprehensive view of tumor heterogeneity. This approach reveals molecular alterations and phenotypic features related to tumor subtypes, potentially identifying therapeutic biomarkers. Many achievements have been made; however, despite continuous advances in proteogenomics-based methodologies, several challenges remain: in particular the limitations in sensitivity and specificity and the lack of optimal study models. This review highlights the impact of proteogenomics on characterizing tumor phenotypes, focusing on the critical challenges and current limitations of its use in different clinical and preclinical models for tumor phenotypic characterization. [ABSTRACT FROM AUTHOR]

The article reports on the public repository towards data of mass spectrometry (MS) imaging. It mentions the dependence of its graphical representation on the normalization of intensity, spatial interpolation and color scale. An overview of the Proteomics Standards Institute and its guidelines is also presented.

Background: Data have accumulated implicating the involvement of oncogenic human papillomaviruses (HPVs) in bronchial carcinogenesis. We recently described the presence of oncogenic HPV transcripts in non-small cell lung cancers. Objective: To investigate the role of oncogenic HPVs in lung carcinogenesis. Material and Methods: The lung cell line A549 stably infected with HPV16E6, HPV16E7 and HPVE6/E7 constructs was used to investigate the protein profile changes associated with the expression of these oncogenes. Replicated two-dimensional gel electrophoresis gels from uninfected and stably HPV16E6-, E7-, and E6/E7-infected A549 cells were compared for changes in protein profile. Protein identification was achieved by peptide mass fingerprinting by MALDI-TOF-MS and nLC-ESI-Q-TOF-MS/MS peptide ladder sequencing. Results: We identified 17 different polypeptides whose average normalized spot intensity was statistically significant (p < 0.05) and differed by 2-fold. Relationships between differentially expressed proteins and the HPV-induced infection mechanism have been clustered by knowledge-base database functional association network analysis. Conclusion: The impact of Hsp27, annexin III, annexin IV, Gp96 and TPT1 on the cellular response mechanism to HPV infection is presented and discussed. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The quantization of glycated isoforms of hemoglobin has been increasingly used in clinical practice in recent years. Glycated hemoglobin is currently considered the most important measurement for long-term control of the glycemic state and it has become a reference tool for the management of diabetes. Glutathionylated hemoglobin is an increasingly clinically relevant covalent adduct of glutathione with β chain of the globin and its concentration has been correlated with oxidative stress. We have developed an innovative technique based on linear mode matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for quantitative analysis of hemoglobin species. This method was applied to the quantification of glycated and glutathionylated hemoglobin. A rigorous comparison was pursued to evaluate the analytical performances in quantifying glycated hemoglobin in comparison to an established high-performance liquid chromatography method. Our results indicated a complete equivalence between the two methods. The same analysis enabled the quantitative determination of the glutathionylated hemoglobin fraction. This isoform was investigated in an adult Italian population (184 individuals, 101 males and 83 females), indicating a bimodal distribution of this species. In fact 65.22% of screened individuals had glutathionylated hemoglobin levels lower than 0.50% while 34.78% had glutathionylated hemoglobin levels higher than 0.50%. A semiautomatic robotic procedure was developed for fast analysis of a large number of samples. This is the first report of a quantitative application of linear MALDI-TOF mass spectrometry for the determination of glutathionylated hemoglobin in blood samples. This method allows fast screening of this hemoglobin isoform, therefore opening the route to explore its specificity and sensitivity as a molecular biomarker. [Copyright &y& Elsevier]

Ependymoma pediatric brain tumor occurs at approximate frequencies of 10–15% in supratentorial and 20–30% in posterior fossa regions. These tumors have an almost selective response to surgery and relative and confirmed resistance to radiotherapy and chemotherapic agents, respectively. Alongside histopathological grading, clinical and treatment evaluation of ependymomas currently consider the tumor localization and the genomic outlined associated molecular subgroups, with the supratentorial and the posterior fossa ependymomas nowadays considered diverse diseases. On these grounds and in trying to better understand the molecular features of these tumors, the present investigation aimed to originally investigate the proteomic profile of pediatric ependymoma tissues of different grade and localization by mass spectrometry platforms to disclose potential distinct protein phenotypes. To this purpose, acid-soluble and acid-insoluble fractions of ependymoma tumor tissues homogenates were analyzed by LC-MS following both the top-down and the shotgun proteomic approaches, respectively, to either investigate the intact proteome or its digested form. The two approaches were complementary in profiling the ependymoma tumor tissues and showed distinguished profiles for supratentorial and posterior fossa ependymomas and for WHO II and III tumor grades. Top-down proteomic analysis revealed statistically significant higher levels of thymosin beta 4, 10 kDa heat shock protein, non-histone chromosomal protein HMG-17, and mono-/uncitrullinated forms ratio of the glial fibrillary acidic protein (GFAP) fragment 388–432 in supratentorial ependymomas—the same GFAP fragment as well as the hemoglobin alpha- and the beta-chain marked grade II with respect to grade III posterior fossa ependymomas. Gene ontology classification of shotgun data of the identified cancer and the non-cancer related proteins disclosed protein elements exclusively marking tumor localization and pathways that were selectively overrepresented. These results, although preliminary, seem consistent with different protein profiles of ependymomas of diverse grade of aggressiveness and brain region development and contributed to enlarging the molecular knowledge of this still enigmatic tumor. [ABSTRACT FROM AUTHOR]

Abstract: Protein profiling of cerebrospinal fluid in Guillain–Barrè syndrome (GBS), an acute and immune-mediated disease affecting the peripheral nervous system, was performed by two-dimensional electrophoresis. Significant modulated spots in GBS patients vs. control groups (a group of multiple sclerosis patients and one of healthy donors) underwent MALDI-TOF/TOF investigation. Inflammation-related proteins, such as vitamin D-binding protein, beta-2 glycoprotein I (ApoH), and a complement component C3 isoform were up-regulated in GBS, whereas transthyretin (the monomer and the dimer forms), apolipoprotein E, albumin and five of its fragments were down-regulated. Then, we used an isoelectric-focusing-dinitrophenylhydrazine-based technique to analyse the extent of carbonylation and, as a result, of oxidative damage of GBS CSF proteome. We observed a major sensitivity to carbonylation for albumin and alpha-glycoprotein in inflammation and a selective increase of reactivity for a glycosylated Fab from an IgM globulin in GBS CSF. Our results add new proteins to candidate CSF features of GBS, and suggest that oxidative stress could contribute to the immunopathological mechanisms in this syndrome. [ABSTRACT FROM AUTHOR]

Abstract: Molecular mass spectrometry (MS) analysis of protein phosphorylation is partially limited by the molecular specie specificity of the analytical responses that might impair both qualitative and quantitative performances. Elemental MS, such as inductively coupled plasma mass spectrometry (ICP-MS) can overcome these drawbacks; in fact, analytical performance is theoretically independent of the molecular structure of a target analyte naturally containing the elements of interest. Nevertheless, isobaric interferences derived from sample matrix and laboratory environment can hinder the quantitative determination of both phosphorus (P) and sulfur (S) as 31P+ and 32S+ by inductively coupled plasma quadrupole mass spectrometry (ICP-QMS) under standard plasma conditions. These interferences may be overcome by quantifying P and S as oxide ions 31P16O+ and 32S16O+, respectively. In this study, we present a systematic investigation on the effect of plasma instrumental conditions on the oxide ion responses by a design of experiment approach for the simultaneous ICP-QMS determination of P and S (31P16O+ and 32S16O+, respectively) in protein samples without the use of dynamic reaction, collision reaction cells or pre-addition of oxygen as reactant gas in the torch. The proposed method was evaluated in terms of limit of detection, limit of quantification, linearity, repeatability, and trueness. Moreover, detection and quantification capabilities of the optimized method were compared to the standard plasma mode for determination of 31P+ and 34S+. Spectral and non-spectral interferences affecting the quantification of 31P+, 31P16O+ and 32S16O+ were also studied. The suitability of inorganic elemental standards for P and S quantification in proteins was assessed. The method was applied to quantify the phosphorylation stoichiometry of commercially available caseins (bovine β-casein, native and dephosphorylated α-casein) and results were confirmed by Matrix Assisted Laser Desorption Ionization Time of Flight MS analysis. We demonstrate that ICP-QMS, by quantifying P and S as oxide ions, was able to accurately calculate the degree of phosphorylation of β-casein and α-casein and to detect specific partial enzymatic dephosphorylation. The collected results might lead to further development of ICP-QMS interfaces optimized for protein phosphorylation studies and for proteomics investigations. [Copyright &y& Elsevier]

Abstract: Background: Mass spectrometry (MS) is becoming the gold standard for biomarker discovery. Several MS-based bioinformatics methods have been proposed for this application, but the divergence of the findings by different research groups on the same MS data suggests that the definition of a reliable method has not been achieved yet. In this work, we propose an integrated software platform, MASCAP, intended for comparative biomarker detection from MALDI-TOF MS data. Results: MASCAP integrates denoising and feature extraction algorithms, which have already shown to provide consistent peaks across mass spectra; furthermore, it relies on statistical analysis and graphical tools to compare the results between groups. The effectiveness in mass spectrum processing is demonstrated using MALDI-TOF data, as well as SELDI-TOF data. The usefulness in detecting potential protein biomarkers is shown comparing MALDI-TOF mass spectra collected from serum and plasma samples belonging to the same clinical population. Conclusions: The analysis approach implemented in MASCAP may simplify biomarker detection, by assisting the recognition of proteomic expression signatures of the disease. A MATLAB implementation of the software and the data used for its validation are available at http://www.unich.it/proteomica/bioinf. [Copyright &y& Elsevier]

Abstract: Background: The development of MALDI Imaging Mass Spectrometry is a promising technique in the investigation of biological molecular repertoire. We have pursued an analytical assessment of this technique in its application to proteome analysis. Methods: A specific statistical method of analysis has been developed to enable data processing in the absence of internal standards, by defining similarity scores. Results: The investigated linear mode MALDI-TOF set-up allows to obtain data variations comprised within the 30% of variation when assaying tissues samples from the same animal, while the 60% of variation was highlighted in the inter-mice series assaying syngenic animals. Conclusions: This analytical assessment represents the first step of a process that should validate the utilisation of this technique in the clinical practice. [Copyright &y& Elsevier]

Food allergy is the disease where the immune system is elicited by antigens in food. Although innocuous for immune-tolerant individuals, an ever-growing number of food allergenic people are being registered worldwide. To date, no treatment to cure food allergy is available and the disease management relies on the careful exclusion of the allergenic food from the diet of the allergic individuals. Great efforts are ongoing to clarify the allergenic mechanisms of the diverse allergenic proteins of food origin, aimed to both designing suitable therapies and for a timely and precise diagnosis of the allergic condition. Among the other omics sciences, mass spectrometry (MS)-based proteomics is gaining a steadily increasing interest by the whole scientific community acknowledged its high versatility. In the present work, the latest proteomics based-studies on allergenic proteins are reviewed to provide guidance on the different MS-based methodologies adopted in the research on food allergens. Our review points to highlight the strengths of the MS-based proteomics and how these have been exploited to address specific research questions. Also, the most common drawbacks encountered in a proteomic study are discussed, providing an overview that helps novel researchers in choosing the more suitable experimental workflow. Wide wealth of knowledge arising from the various MS-based proteomic investigations is improving our understanding of food allergy through molecular characterization of food allergens. The present work reviews the key aspects to be evaluated while investigating food allergens by means of MS-based proteomics and provide guidance to the novel research groups approaching to the fascinating world of MS-based food allergens detection. Unlabelled Image • Food allergy represents a steadily increasing public health threat. • MS-based proteomics enables deep characterization of food allergens. • MS-proteomics unequivocally identifies multiple allergens per run. • Harmonization between allergen quantitation methods is needed. • Exploring sensibility is the next challenge of MS-based food allergen analysis. [ABSTRACT FROM AUTHOR]