Sputum proteomics in lung disorders.

Lung diseases affect pulmonary and respiratory function and are caused by bacterial viral and fungal infection as well as environmental factors. Unfortunately, symptom overlap between various pulmonary diseases often prevents clear differentiation and uncertain diagnosis. Accordingly, identification of specific markers of inflammatory activity in early disease stage could potential unveil the intrinsic molecular mechanisms of the underlying pathology. Proteomic studies aimed at understanding the genetic/environmental contributions to the development and progression of lung diseases represent a promising approach for diagnosis and treatment. The fluid phase of sputum represents a rich protein source and is frequently used in these studies. This chapter addresses causes of lung disorders, sputum composition, collection and processing as well as the clinical significance and challenges associated with the presence of interfering factors. Basics of proteomics and mass spectrometry are also described, together with the analytical approaches to investigate the sputum proteome. Finally, we explore the application of sputum proteomics in severe lung disorders including COVID-19 infection, chronic obstructive pulmonary disease, asthma, cystic fibrosis, lung cancer and tuberculosis.

Chemotherapy-Induced Changes in Plasma Amino Acids and Lipid Oxidation of Resected Patients with Colorectal Cancer: A Background for Future Studies.

Previous studies have documented that FOLFOX and XELOX therapies negatively impact the metabolism of skeletal muscle and extra-muscle districts. This pilot study tested whether three-month FOLFOX or XELOX therapy produced changes in plasma amino acid levels (PAAL) (an estimation of whole-body amino acid metabolism) and in plasma levels of malondialdehyde (MDA), a marker of lipid hyper oxidation. Fourteen ambulatory, resected patients with colorectal cancer scheduled to receive FOLFOX (n = 9) or XELOX (n = 5) therapy, after overnight fasting, underwent peripheral venous blood sampling, to determine PAAL and MDA before, during, and at the end of three-month therapy. Fifteen healthy matched subjects (controls) only underwent measures of PAAL at baseline. The results showed changes in 87.5% of plasma essential amino acids (EAAs) and 38.4% of non-EAAs in patients treated with FOLFOX or XELOX. These changes in EAAs occurred in two opposite directions: EAAs decreased with FOLFOX and increased or did not decrease with XELOX (interactions: from p = 0.034 to p = 0.003). Baseline plasma MDA levels in both FOLFOX and XELOX patients were above the normal range of values, and increased, albeit not significantly, during therapy. In conclusion, three-month FOLFOX or XELOX therapy affected plasma EAAs differently but not the baseline MDA levels, which were already high.

CD73-Adenosinergic Axis Mediates the Protective Effect of Extracellular Vesicles Derived from Mesenchymal Stromal Cells on Ischemic Renal Damage in a Rat Model of Donation after Circulatory Death.

We propose a new organ-conditioning strategy based on mesenchymal stromal cell (MSCs)/extracellular vesicle (EVs) delivery during hypothermic perfusion. MSCs/EVs marker CD73 is present on renal proximal tubular cells, and it protects against renal ischemia-reperfusion injury by converting adenosine monophosphate into adenosine (ADO). In this study, after checking if CD73-silenced EVs (EVsi) would impact in vitro tubular-cell proliferation, we perfused kidneys of a rat model of donation after circulatory death, with Belzer solution (BS) alone, BS supplemented with MSCs, EVs, or EVsi. The ADO and ATP levels were measured in the effluents and tissues. Global renal ischemic damage score (GRS), and tubular cell proliferation index (IPT) were evaluated in the tissue. EVsi did not induce cell proliferation in vitro. Ex vivo kidneys perfused with BS or BS + EVsi showed the worst GRS and higher effluent ADO levels than the MSC- and EV-perfused kidneys. In the EV-perfused kidneys, the tissue and effluent ATP levels and IPT were the highest, but not if CD73 was silenced. Tissue ATP content was positively correlated with tissue ADO content and negatively correlated with effluent ADO level in all groups. In conclusion, kidney conditioning with EVs protects against ischemic damage by activating the CD73/ADO system.

Proteomic Analysis of Human Sputum for the Diagnosis of Lung Disorders: Where Are We Today?

The identification of markers of inflammatory activity at the early stages of pulmonary diseases which share common characteristics that prevent their clear differentiation is of great significance to avoid misdiagnosis, and to understand the intrinsic molecular mechanism of the disorder. The combination of electrophoretic/chromatographic methods with mass spectrometry is currently a promising approach for the identification of candidate biomarkers of a disease. Since the fluid phase of sputum is a rich source of proteins which could provide an early diagnosis of specific lung disorders, it is frequently used in these studies. This report focuses on the state-of-the-art of the application, over the last ten years (2011-2021), of sputum proteomics in the investigation of severe lung disorders such as COPD; asthma; cystic fibrosis; lung cancer and those caused by COVID-19 infection. Analysis of the complete set of proteins found in sputum of patients affected by these disorders has allowed the identification of proteins whose levels change in response to the organism's condition. Understanding proteome dynamism may help in associating these proteins with alterations in the physiology or progression of diseases investigated.

GPR120 prevents colorectal adenocarcinoma progression by sustaining the mucosal barrier integrity.

GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by ω-3 Polyunsaturated Fatty Acids (PUFAs), and expressed in many cell types. Its role in the context of colorectal cancer (CRC) is still puzzling with many controversial evidences. Here, we explored the involvement of epithelial GPR120 in the CRC development. Both in vitro and in vivo experiments were conducted to mimic the conditional deletion of the receptor from gut epithelium. Intestinal permeability and integrity of mucus layer were assessed by using Evans blue dye and immunofluorescence for MUC-2 protein, respectively. Microbiota composition, presence of lipid mediators and short chain fatty acids were analyzed in the stools of conditional GPR120 and wild type (WT) mice. Incidence and grade of tumors were evaluated in all groups of mice before and after colitis-associated cancer. Finally, GPR120 expression was analyzed in 9 human normal tissues, 9 adenomas, and 17 primary adenocarcinomas. Our work for the first time highlights the role of the receptor in the progression of colorectal cancer. We observed that the loss of epithelial GPR120 in the gut results into increased intestinal permeability, microbiota translocation and dysbiosis, which turns into hyperproliferation of epithelial cells, likely through the activation of ß -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. these results demonstrate that the epithelial GPR120 receptor is essential to maintain the mucosal barrier integrity and to prevent CRC developing. Therefore, our data pave the way to GPR120 as an useful marker for the phenotypic characterization of CRC lesions and as new potential target for CRC prevention.

A Shotgun Proteomic Platform for a Global Mapping of Lymphoblastoid Cells to Gain Insight into Nasu-Hakola Disease.

Nasu-Hakola Disease (NHD) is a recessively inherited systemic leukodystrophy disorder characterized by a combination of frontotemporal presenile dementia and lytic bone lesions. NHD is known to be genetically related to a structural defect of TREM2 and DAP12, two genes that encode for different subunits of the membrane receptor signaling complex expressed by microglia and osteoclast cells. Because of its rarity, molecular or proteomic studies on this disorder are absent or scarce, only case reports based on neuropsychological and genetic tests being reported. In light of this, the aim of this paper is to provide evidence on the potential of a label-free proteomic platform based on the Multidimensional Protein Identification Technology (MudPIT), combined with in-house software and on-line bioinformatics tools, to characterize the protein expression trends and the most involved pathways in NHD. The application of this approach on the Lymphoblastoid cells from a family composed of individuals affected by NHD, healthy carriers and control subjects allowed for the identification of about 3000 distinct proteins within the three analyzed groups, among which proteins anomalous to each category were identified. Of note, several differentially expressed proteins were associated with neurodegenerative processes. Moreover, the protein networks highlighted some molecular pathways that may be involved in the onset or progression of this rare frontotemporal disorder. Therefore, this fully automated MudPIT platform which allowed, for the first time, the generation of the whole protein profile of Lymphoblastoid cells from Nasu-Hakola subjects, could be a valid approach for the investigation of similar neurodegenerative diseases.

Differences and Effects of Metabolic Fate of Individual Amino Acid Loss in High-Efficiency Hemodialysis and Hemodiafiltration.

OBJECTIVE: The objective of the study was to quantify the loss and arterial blood concentration of the three main classes of amino acids (AAs)-nonessential amino acids (NEAAs), essential amino acids (EAAs), and branched-chain amino acids-as resulting from high-efficiency hemodialysis (HED) and hemodiafiltration (HDF). We moreover aimed to identify the different fates and metabolic effects manifested in patients undergoing hemodialysis and the consequences on body composition and influence of nutritional decline into protein energy wasting. DESIGN AND METHODS: Identical dialysis monitors, membranes, and dialysate/infusate were used to ensure consistency. Ten patients were recruited and randomized to receive treatment with on-line modern HED and HDF. Arterial plasma concentrations of individual AAs were compared in healthy volunteers and patients undergoing hemodialysis, and AA levels outflowing from the dialyzer were evaluated. Baseline AA plasma levels of patients undergoing hemodialysis were compared with findings obtained 1 year later. RESULTS: A severe loss of AA with HED/HDF was confirmed: a marked loss of total AAs (5 g/session) was detected, corresponding to more than 65% of all AAs. With regard to individual AAs, glutamine displayed a consistent increase (+150%), whereas all other AAs decreased after 12 months of HD/HDF. Only a few AAs, such as proline, cysteine, and histidine maintained normal levels. The most severe metabolic consequences may result from losses of EAAs such as valine, leucine, and histidine and from NEAAs including proline, cysteine, and glutamic acid eliciting the onset of hypercatabolism threatening muscle mass loss. CONCLUSION: Dialysis losses, together with the effect of chronic uremia, resulted in a reduction of fundamental EAAs and NEAAs, which progressively led our patients after 12 months to a deterioration of lean mass toward sarcopenia. Therefore, the reintroduction of a correctly balanced AA supplementation in patients undergoing HD to prevent or halt decline of hypercatabolism into cachexia is recommended.

The Hip Functional Retrieval after Elective Surgery May Be Enhanced by Supplemented Essential Amino Acids.

It is not known whether postsurgery systemic inflammation and plasma amino acid abnormalities are still present during rehabilitation of individuals after elective hip arthroplasty (EHA). Sixty subjects (36 females; age 66.58 ± 8.37 years) were randomized to receive 14-day oral EAAs (8 g/day) or a placebo (maltodextrin). At admission to and discharge from the rehabilitation center, serum C-reactive protein (CRP) and venous plasma amino acid concentrations were determined. Post-EHA hip function was evaluated by Harris hip score (HHS) test. Ten matched healthy subjects served as controls. At baseline, all patients had high CRP levels, considerable reduction in several amino acids, and severely reduced hip function (HHS 40.78 ± 2.70 scores). After treatment, inflammation decreased both in the EAA group and in the placebo group. Only EAA patients significantly improved their levels of glycine, alanine, tyrosine, and total amino acids. In addition, they enhanced the rate of hip function recovery (HHS) (from baseline 41.8 ± 1.15 to 76.37 ± 6.6 versus baseline 39.78 ± 4.89 to 70.0 ± 7.1 in placebo one; p = 0.006). The study documents the persistence of inflammation and plasma amino acid abnormalities in post-EHA rehabilitation phase. EAAs enhance hip function retrieval and improve plasma amino acid abnormalities.

Protein networks in induced sputum from smokers and COPD patients.

RATIONALE: Subtypes of cigarette smoke-induced disease affect different lung structures and may have distinct pathophysiological mechanisms. OBJECTIVE: To determine if proteomic classification of the cellular and vascular origins of sputum proteins can characterize these mechanisms and phenotypes. SUBJECTS AND METHODS: Individual sputum specimens from lifelong nonsmokers (n=7) and smokers with normal lung function (n=13), mucous hypersecretion with normal lung function (n=11), obstructed airflow without emphysema (n=15), and obstruction plus emphysema (n=10) were assessed with mass spectrometry. Data reduction, logarithmic transformation of spectral counts, and Cytoscape network-interaction analysis were performed. The original 203 proteins were reduced to the most informative 50. Sources were secretory dimeric IgA, submucosal gland serous and mucous cells, goblet and other epithelial cells, and vascular permeability. RESULTS: Epithelial proteins discriminated nonsmokers from smokers. Mucin 5AC was elevated in healthy smokers and chronic bronchitis, suggesting a continuum with the severity of hypersecretion determined by mechanisms of goblet-cell hyperplasia. Obstructed airflow was correlated with glandular proteins and lower levels of Ig joining chain compared to other groups. Emphysema subjects' sputum was unique, with high plasma proteins and components of neutrophil extracellular traps, such as histones and defensins. In contrast, defensins were correlated with epithelial proteins in all other groups. Protein-network interactions were unique to each group. CONCLUSION: The proteomes were interpreted as complex "biosignatures" that suggest distinct pathophysiological mechanisms for mucin 5AC hypersecretion, airflow obstruction, and inflammatory emphysema phenotypes. Proteomic phenotyping may improve genotyping studies by selecting more homogeneous study groups. Each phenotype may require its own mechanistically based diagnostic, risk-assessment, drug- and other treatment algorithms.

Efflux-mediated resistance to a benzothiadiazol derivative effective against Burkholderia cenocepacia.

Burkholderia cenocepacia is a major concern for people suffering from cystic fibrosis as it contributes to serious respiratory tract infections. The lack of drugs effective against this opportunistic pathogen, along with the high level of resistance to multiple antibiotics, render the treatment of these infections particularly difficult. Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109), with a bactericidal effect and a minimal inhibitory concentration (MIC) of 8 µg/ml against B. cenocepacia, is described. The compound is not cytotoxic and effective against B. cenocepacia clinical isolates and members of all the known B. cepacia complex species. Spontaneous mutants resistant to 10126109 were isolated and mutations in the MerR transcriptional regulator BCAM1948 were identified. In this way, a mechanism of resistance to this new molecule was described, which relies on the overexpression of the RND-9 efflux pump. Indeed, rnd-9 overexpression was confirmed by quantitative reverse transcription PCR, and RND-9 was identified in the membrane fractions of the mutant strains. Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

Conductivity in Exhaled Breath Condensate from Subjects with Emphysema and Type ZZ alpha-1-Antitrypsin Deficiency.

The assessment of biomarkers in biological samples from the lung has long been employed. Upon cooling water vapor present in exhaled breath, variable amounts of droplets of condensate (EBC) containing volatile and non-volatile compounds may be easily and non-invasively obtained from patients of any age.Objective of the present study was to compare the level of EBC conductivity determined for cohorts of individuals with different inflammatory lung disorders with that of healthy never-smoking individuals.The conductivity in EBC of PiZZ-Alpha-1-antitrypsin deficiency patients with a diagnosis of emphysema (PiZZ-AATD) was 3 fold lower than in spouse controls (54.5 ± 11.6 vs 165.3 ± 10.7 µS/cm). Non-PiZZ emphysema patients had conductivity in EBC of 59.6 ± 5.8 µS/cm and patients with sarcoidosis without airflow obstruction had EBC conductivity of 178,8 ± 6,2 µS/cm, not significantly different (p = 0.5) from healthy controls. Conductivity in serial EBC samples from patients with PiZZ-AATD emphysema and healthy controls was stable in 6 different samples collected over a period of 14 months. We conclude that conductivity values in EBC can be used as a correction factor for dilution of non-volatile components in EBC.

Proteomic analysis of lymphoblastoid cells from Nasu-Hakola patients: a step forward in our understanding of this neurodegenerative disorder.

Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer's disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses.

Is stroke rehabilitation a metabolic problem?

BACKGROUND: This study looks at the impact of inflammation during the rehabilitation stage of strokes and its effect on neuro-functional recovery. METHODS: This study investigated 94 patients suffering from strokes and admitted to rehabilitation. Anthropometric characteristics, serum proteins and inflammatory markers, plasma amino acids and neurofunction were all assessed. RESULTS: 55.3% patients had an inflammatory status (Interleukin-6 = 19.24 ± 23.01 pg ml⁻¹ vs. 4.1 ± 1.6 pg ml⁻¹ for non-inflamed subjects (p < 0.001). Inflammation was positively linked to positive proteins (alpha-1 globulin, p < 0.02) and negatively linked to negative proteins (albumin, p < 0.02; prealbumin, p < 0.01; transferrin, p < 0.05) of the acute-phase response. Inflammation was associated with low plasma concentrations of total amino acids. For the multiple logistic regression analysis, albumin (p < 0.001) and body weight maintenance (p < 0.001) were independent predictors of patient functional independence. Inflammation in dysphagic stroke (31.9%) patients was associated with more accentuated disability compared to non-inflamed dysphagics. The serum positive reactant alpha 1 globulin was the most powerful predictor of dysphagia severity (p < 0.001). At discharge, dysphagia improvement was associated with improved acute-phase negative proteins. CONCLUSIONS: An inflammatory status may persist for most patients with strokes during the rehabiliation stage of the disease, its prevalence being higher in dysphagic compared to non-dysphagic subjects. The improvement in circulating albumin and body weight maintenance are predictors of neuro-function, even in dysphagic subjects.

Profiling the proteome of exhaled breath condensate in healthy smokers and COPD patients by LC-MS/MS.

Three pools of exhaled breath condensate (EBC) from non-smokers plus healthy smokers (NS + HS, n = 45); chronic obstructive pulmonary disease (COPD) without emphysema (COPD, n = 15) and subjects with pulmonary emphysema associated with α(1)-antitrypsin deficiency (AATD, n = 23) were used for an exploratory proteomic study aimed at generating fingerprints of these groups that can be used in future pathophysiological and perhaps even clinical research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the platform applied for this hypothesis-free investigation. Analysis of pooled specimens resulted in the production of a "fingerprint" made of 44 proteins for NS/HS; 17 for COPD and 15 for the group of AATD subjects. Several inflammatory cytokines (IL-1α, IL-1ß, IL-2; IL-12, α and ß subunits, IL-15, interferon α and γ, tumor necrosis factor α); Type I and II cytokeratins; two SP-A isoforms; Calgranulin A and B and α1-antitrypsin were detected and validated through the use of surface enhanced laser-desorption ionization mass spectrometry (SELDI-MS) and/or by Western blot (WB) analysis. These results are the prelude of quantitative studies aimed at identifying which of these proteins hold promise as identifiers of differences that could distinguish healthy subjects from patients.

The developmental neurotoxicity study of platinum compounds. Effects of cisplatin versus a novel Pt(II) complex on rat cerebellum.

In the field of experimental oncology, many efforts are being carried out to search new platinum-based drugs overcoming the CNS toxicity and drug resistance. One of the adopted strategies is the synthesis of platinum compounds able to form Pt-DNA adducts different from the cisplatin ones or to react with other subcellular targets. In this context a novel Pt(II) complex, [Pt(O,O'-acac)(γ-acac)(DMS)](PtAcacDMS), was synthesized which reacts preferentially with protein thiols or thioethers. In this work we investigated the in vivo effects of cisplatin and PtAcacDMS on normal development. Moreover, to verify the dose-dependence of the effects, different groups of animals were treated with 5 µg/g or 10 µg/g body weight of cisPt and PtAcacDMS. We have focused our attention on the cerebellum because it provides a useful model system to evaluate the outcomes of perinatal treatment with chemotherapeutic agents on key CNS developmental processes such as neural cells proliferation, migration and differentiation. We have demonstrated the ability of both cisPt and PtAcacDMS to reach the brain tissue once injected. The brain platinum content after PtAcacDMS treatment was notably higher (approximately 4-fold as much) than after cisPt. The platinum accumulation in the brain was still considerable 7 days after PtAcacDMS administration. However, compared with cisplatin, PtAcacDMS induces less severe changes on fundamental events of neuroarchitecture development, such as no high apoptotic events, less altered granule cell migration and Purkinje cell dendrite growth, suggesting a low neurotoxicity of this new Pt complex for normal CNS. The mild damages could be attributable to the different subcellular target of this compound as well as to a greater efficiency of the cell repair system to recognize the drug-target adducts and to repair them. Together with the previously demonstrated antineoplastic effectiveness in vitro, the findings here reported suggest PtAcacDMS as a potential alternative to cisplatin indicating, at the same time, that the choice of platinum compounds with new subcellular targets could be a strategy to prevent neurotoxicity induced by cisplatin and overcome drug resistance induced by mutations in the intrinsic apoptotic pathway.

Alveolar inflammation in cystic fibrosis.

BACKGROUND: In infected lungs of the cystic fibrosis (CF) patients, opportunistic pathogens and mutated cystic fibrosis transmembrane conductance regulator protein (CFTR) contribute to chronic airway inflammation that is characterized by neutrophil/macrophage infiltration, cytokine release and ceramide accumulation. We sought to investigate CF lung inflammation in the alveoli. METHODS: Lung tissue from 14 CF patients and four healthy individuals was analyzed for numbers of effector cells, elastin and collagen concentrations, inflammatory markers and density of Pseudomonas aeruginosa. Additionally, desmosine and isodesmosine concentrations were determined in 52 urine specimens from CF patients to estimate the burden of elastase activities in respiratory secretions. RESULTS: Elastin concentration was significantly decreased and collagen significantly increased in CF alveolar tissues as compared to age-matched, healthy individuals. Elastin split products were significantly increased in urine samples from patients with CF and correlated inversely with age, indicating local tissue remodelling due to elastin degradation by unopposed proteolytic enzymes. Alveolar inflammation was also characterized by a significant cell infiltration of neutrophils, macrophages and T cells, extensive nuclear factor-kappaB and insulin-like growth factor-1 activation in various cell types and increased intercellular adhesion molecule-1 expression, and increased numbers of myofibroblasts. Additionally, ceramide accumulated in type II alveolar epithelial cells, lacking CFTR. P. aeruginosa organisms were rarely present in inflamed alveoli. CONCLUSIONS: Chronic inflammation and remodeling is present in alveolar tissues of the CF lung and needs to be addressed by anti-inflammatory therapies.

A rapid MEKC method for the simultaneous determination of creatinine, 1- and 3-methylhistidine in human urine.

The present report describes the use of MEKC in the presence of 35 mM sodium tetraborate, pH 9.0, containing 60 mM SDS for the complete separation and identification of creatinine, 1-methylhistidine (1-MeH) and 3-MeH in human urine. Their simultaneous quantification in urine of healthy controls and subjects submitted to elective surgery to their lower limbs allowed to use 3-MeH as a reliable measure of skeletal protein breakdown. Simplicity, sensitivity and low running costs are the main advantages of this method that is suitable for the routine analysis in clinical laboratories of a large number of samples per day.

2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Y cell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOF-MS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS.

Optimizing separation efficiency of 2-DE procedures for visualization of different superoxide dismutase forms in a cellular model of amyotrophic lateral sclerosis.

Neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease (PD) have been associated with increased production of reactive oxygen species. In AD and PD patients, superoxide dismutase (SOD1) was also indicated as a major target of oxidative damage. In particular, in brain tissue of these patients, different SOD1 isoforms have been identified, although their functional role still remains to be elucidated. In the light of the possibility that different SOD1 entities could be expressed also in other neurodegenerative disorders, as a sort of unifying event with AD and PD, we have investigated amyotrophic lateral sclerosis (ALS) using human neuroblastoma SH-SY5Y cells with mutated SOD1 gene H46R as cellular model. 2-DE using a narrow-range IPG 4-7 strips in the first dimension and linear 15% SDS-PAGE in the second allowed to separate different SOD1 spots. MALDI-TOF MS and CapLC-MS/MS have been used for their complete identification. This is the first report in which the presence of SOD1 (iso) forms in a cellular model of ALS has been evidenced.

Protein expression in sputum of smokers and chronic obstructive pulmonary disease patients: a pilot study by CapLC-ESI-Q-TOF.

The current report describes the use of CapLC-ESI-Q/TOF-MS for investigating the proteome profiles of hypertonic saline-induced sputum samples from 56 smokers. The severity of their lung disease ranged from normal (healthy smokers) to chronic bronchitis, chronic obstructive pulmonary disease (COPD), and COPD with emphysema. This pilot study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype and cigarette smoking illness severity. A total of 203 unique proteins were identified. These may represent the most highly expressed proteins in induced sputum. Our results provide evidence that different proteins are expressed, as the disease progresses from health to more advanced stages, and support our contention that a proteomic approach would be beneficial in discovering selective molecules linked to specific COPD stages.