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Tumor extracellular matrix (ECM) plays a pivotal role in outcome of breast cancer (BC) patients. Overespression of 58 genes, encoding 43 structural ECM proteins, has been identified to determine a specific cluster of BC with accelerated metastatic potential only in the undifferentiated (grade III) phenotype. The scope of this study was to characterize protein repertoire able to predict patient outcome in BC according to ECM gene expression pattern and histological grade. The differential proteomic analysis has been based on 2D-DIGE, MALDI-MS, bioinformatics and immunoblotting. Results suggested a relationship among ECM remodeling, signal mechanotransduction and metabolic rewiring in BCs characterized by a specific mRNA ECM signature and identified a set of dysregulated proteins characteristic of hormone receptors expression as fibrinogen beta chain (FGB), collagen alpha-1 (VI) chain (COL6A1) and alpha-1B-glycoprotein (A1BG). Furthermore, in triple negative tumors (TN) with ECM signature, the FGG and ?5?1/?v?3 integrins increased whereas detyrosinated alpha-tubulin, and mimecan (OGN) decreased leading to unorganized integrin presentation involving focal adhesion kinase (FAK), activation of Rho GTPases associated to epithelial mesenchymal transition. In hormone receptors negative BCs characterized by a specific ECM gene cluster, the differentially regulated proteins, identified in the present study,can be potentially relevant to predict patient's outcome.

Muscle represents a large fraction of the human body mass. It is an extremely heterogeneous tissue featuring in its contractile structure various proportions of heavy- and light-chain slow type 1 and fast type 2A and 2X myosins, actins, tropomyosins, and troponin complexes as well as metabolic proteins (enzymes and most of the players of the so-called excitation-transcription coupling). Muscle is characterized by wide plasticity, i.e. capacity to adjust size and functional properties in response to endogenous and exogenous influences. Over the last decade, proteomics has become a crucial technique for the assessment of muscle at the molecular level and the investigation of its functional changes. Advantages and shortcomings of recent techniques for muscle proteome analysis are discussed. Data from differential proteomics applied to healthy individuals in normal and unusual environments (hypoxia, cold), in exercise, immobilization, ageing and to patients with neuromuscular hereditary disorders (NMD's), inclusion bodies myositis and insulin resistance are summarized, critically discussed and, when required, compared with homologous data from pertinent animal models. The advantages as well as the limits of proteomics in view of the identification of new biomarkers are evaluated.

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1±), energy balance (AMPK), mitochondrial biogenesis (PGC-1±), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1± stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1±, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.

The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (±-cyano-4-hydroxycinnamic acid and 2',4'-dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2',4'-dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (

Recent advances in stem cell biology have demonstrated that terminally differentiated adult cells can be induced to de-differentiate into progenitor cells (induced stem cells) upon proper stimuli. This has been achieved by the induced expression of key regulatory genes by retro- or lenti-viral systems. On the other hand, synthetic "small molecules" can also induce de-differentiation and may represent a potentially safer approach as compared with genetic manipulation. Along this line, a synthetic purine called "reversine" has been shown to induce the de-differentiation of fibroblasts into mesenchymal stem-cell-like progenitors, which can be successively induced to differentiate into skeletal muscle, smooth muscle and bone cells. The mechanism whereby reversine is able to achieve de-differentiation has yet to be clarified. In this context, we defined the protein changes induced by reversine treatment in murine fibroblasts by 2-D difference gel electrophoresis, coupled with MS. Proteins involved in cytoskeletal and cell shape remodeling, RNA export, degradation, folding, stress control and ATP production were found to be remarkably changed after reversine treatment. Ingenuity pathway analysis (IPA) predicted that these protein pattern changes enabled to propose that about 40 proteins might be associated to several biological functional networks, including cellular assembly, cell signaling and cell death. Altogether our data confirm the intrinsic complexity of the de-differentiation process induced by reversine and suggest more selected approaches to investigate the action mechanism of this small molecule.

Ageing induces a progressive morphological change and functional decline in muscles and in nerves. Light and electron microscopy, 2-D DIGE and MS, were applied to profile the qualitative and quantitative differences in the proteome and morphology of rat gastrocnemius muscle and sciatic nerve, in healthy 22-month-old rats. At muscle level, morphological changes are associated to fibre atrophy accompanied by myofibrillar loss and degeneration, disappearance of sarcomeres and sarcoplasmic reticulum dilatation, internal migration of nuclei, longitudinal fibre splitting, increment of subsarcolemmal mitochondria aggregates and increment of lipofuscin granules. Sciatic nerve shows myelin abnormalities like enfoldings, invaginations, onion bulbs, breakdowns and side axonal atrophy. Proteomic analysis identified changes correlated to morphological abnormalities in metabolic, contractile and cytoskeletal proteins, deregulation of iron homeostasis, change of Ca2+ balance and stress response proteins, accompanied by a deregulation of myelin membrane adhesion protein and proteins regulating the neuronal caliber. By comparing proteomic results from the two tissues, 16 protein isoforms showed the same up and down regulation trend suggesting that there are changes implying a general process which may act as a signal event of degeneration. Only enolase and tropomyosin 1 were differentially expressed in the tissues.

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7-9 days exposure to 4559 m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre- and post-exposure, in ten young subjects, by 2-D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7-9 days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p

We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the expression of contractile and regulatory proteins in human vastus lateralis and deltoideus muscles, in order to understand protein turnover and isoform switching in muscles with the same fiber-type composition but different functional properties. We demonstrate a two- to six-fold overexpression of enzymes associated with glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and substrate transport in vastus lateralis compared to deltoideus. Expression levels of contractile protein isoforms correlated to the proportion of slow-twitch fibers in deltoideus compared to vastus lateralis are consistent with the different contractile properties of the two muscles. Two proteins involved in free radical homeostasis were differentially expressed, suggesting a direct relationship between radical scavenging and the muscle function. The application of 2-DE and MS to studies of muscle physiology thus offers a more comprehensive assessment of the molecular determinants of muscle function than traditional approaches.

Two compounds, derivatives of 1,4-diazobicyclo[2,2,2]octane (DABCO), have been evaluated as potential quenchers of silanol interactions with peptides and proteins during their capillary zone electrophoresis (CZE) separations. They are: 1-(4-iodobutyl)4-aza-1-azoniabicyclo[2,2,2]octane iodide (M7C4I) and 1,4-didecyl-1,4-diazoniabicyclo [2,2,2]octane dibromide (C10M7C10). The first compound is known to react with the wall, by forming a covalent bond via alkylation of silanols. On the contrary, the second one (C10M7C10) can only loosely interact with silica due to lack of reactive iodine and to a much too short distance (a C(2)) between the two quaternary nitrogens. Very good peptide maps of protein digests can be obtained in isoelectric glutamic acid (Glu) buffer, at pH 3.52 by utilizing the M7C4I. However, in the case of total tissue extracts, excellent resolution is obtained only with the first eluting part of the analyte spectrum (i.e., peptides and smaller proteins). With larger proteins, interaction with the wall and loss of resolution is experienced. When using the C10M7C10, good resolution of di- and tripeptides is obtained, while a loss of resolution is observed with entire protein digest. M7C4I does not seem to interact with the peptide/protein analytes, and simply repels them from the wall via its positive charges; it is believed that disalt (C10M7C10) acts by interacting with the same compounds, possibly by forming micelles in solution.

The present mini-review summarizes the experience gathered by our group in developing different classes of novel quaternarized heterocyclic compounds able to modulate and reverse the electroendoosmotic flow (EOF) in a most peculiar manner. The first class comprises mono-salt compounds, with the determinant omega-iodoalkyl chains of different lengths (typically C4-C8), able to be adsorbed by silicas, at alkaline pH, and spontaneously alkylate ionised silanols, thus becoming covalently affixed to it. The second class is constituted by di-salt compounds, attached at the termini of an alkyl chain of variable lengths (here too, typically, C4-C8). This second class is unable to bind covalently silica surfaces, although, in thin-layer chromatography, it exhibits an extraordinary affinity for silica beads, contrary to the first one. On the basis of the strikingly different behaviour, structural rules are derived for the minimum requirements for general classes of amines to bind to silica walls and modify EOF. For compounds unable to bind covalently to the wall, the most important structural motif is two quaternary nitrogens spaced apart by a C4 chain: this seems to be the average distance (i.e., 0.8 nm) between two adjacent, ionized silanols for a snug fit. The other structural binding motif is the "hydrophobic decoration", i.e., the ratio of charged groups to alkyl residue in the various amines; amines with high levels of such alkane groups (i.e., with higher hydrophobicity), seem to bind more tenaciously to the wall, probably due to hydrophobic interaction not to the wall but among the amine derivatives themselves, when carpeting the silica.