Effects of cigarette smoking on the human urinary proteome

In this pilot study we used a proteomic approach to compare the urinary protein patterns of healthy smokers and non-smokers. Proteins were resolved by two-dimensional gel electrophoresis and identified by mass spectrometry. The relative abundance of three inflammatory proteins (S100A8, inter-alpha-trypsin inhibitor heavy chain 4, CD59) and that of two isoforms of pancreatic alpha amylase was significantly higher in smokers. Zinc-alpha-2-glycoprotein was the only protein down-regulated in smokers. Its abundance was significantly correlated with urinary glucocorticoids. Most of the proteins identified may be non-specific biomarkers of tobacco effects, since they are involved in inflammatory responses associated with several diseases. Of greater interest are the changes in abundance of pancreatic alpha amylase and zinc-alpha-2-glycoprotein, which after proper validation, might be candidate biomarkers of diseases resulting from exposure to tobacco smoke. The data also show for the first time that smoking can affect the expression profile of urinary proteins.     

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.     

Proteomic study of human bronchoalveolar lavage fluids from smokers with chronic obstructive pulmonary disease by combining surface-enhanced laser desorption/ionization-mass spectrometry profiling with mass spectrometric protein identification

Bronchoalveolar lavage fluid (BALF) is an important diagnostic source to investigate cellular and molecular changes in the course of lung disorders. The pattern of soluble proteins in BALF obtained from patients at different stages of respiratory disorders may provide deeper insights in the molecular mechanisms of the disease. We used surface-enhanced laser desorption/ionization mass spectrometry (MS) for differential protein display combined with reversed-phase chromatography and subsequent matrix-assisted laser desorption/ionization-MS or nanoliquid chromatography MS/MS analysis for protein identification to compare the protein pattern of BALF samples obtained from ten smokers suffering from chronic obstructive pulmonary disease (COPD), eight clinically asymptomatic smokers, and eight nonsmokers without pulmonary disease. In this context, we were able to identify small proteins and peptides, either differentially expressed or secreted in the course of COPD or in a direct response to cigarette smoke. The concentrations of neutrophil defensins 1 and 2, S100A8 (calgranulin A), and S100A9 (calgranulin B) were elevated in BALFs of smokers with COPD when compared to asymptomatic smokers. Increased concentrations in S100A8 (Calgranulin A), salivary proline-rich peptide P-C, and lysozyme C were detected in BALFs of asymptomatic smokers when compared to nonsmokers, whereas salivary proline-rich peptide P-D and Clara cell phospholipid-binding protein (CC10) were reduced in their concentration. The identified proteins and peptides might be useful in the future as diagnostic markers for smoke-induced lung irritations and COPD.     

Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers

Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.     

Increased plasma concentrations of C9, C1-inhibitor and alpha 1-protease inhibitor associated with cigarette smoking

The association between various parameters of acute and chronic smoking status and plasma levels of three proteins, C9, C1-inhibitor (C1-INH) and alpha 1-protease inhibitor (alpha 1-PI) were determined for 49 male cigarette smokers and 49 age-matched nonsmokers (mean age = 32.2 years). The mean number of cigarettes smoked was 28.7 per day while the cumulative consumption was only 18.1 pack-years. Plasma levels of all three proteins were significantly higher in the smokers than nonsmokers. Plasma C9 and alpha 1-PI concentrations correlated with cumulative cigarette consumption and plasma nicotine concentrations. While C1-INH concentration did not correlate with either cumulative cigarette consumption or plasma nicotine concentration, it correlated significantly with serum thiocyanate concentration. No consistent correlation was found between plasma concentration of these proteins and parameters of pulmonary function.     

葱蝇非滞育与夏滞育蛹蛋白的双向凝胶电泳比较分析

【目的】比较分析葱蝇Delia antiqua非滞育与夏滞育蛹的蛋白表达差异, 为进一步揭示昆虫滞育的分子调控机理和昆虫防治提供理论基础。【方法】以非滞育和夏滞育的蛹为材料, 提取总蛋白; 进行双向凝胶电泳和凝胶图像分析, 对并差异蛋白质进行MALDI-TOF MS质谱鉴定, 获得该点的质量指纹图谱; 利用MASCOT软件在NCBI和SWISS PORT蛋白质数据库中进行搜索鉴定。【结果】非滞育和夏滞育蛹的蛋白表达存在显著差异。通过质谱鉴定和生物信息学分析, 13个差异表达的蛋白质分别为胶原蛋白、 纺锤丝组装非正常蛋白6(SAS6)、 5,10-亚甲基四氢叶酸合成酶(MTHFS)、 Bnb蛋白(bangles and beads)及其他功能未知蛋白。【结论】葱蝇在夏滞育时期, 蛹体内的某些蛋白被上调或下调表达。本研究所鉴定的蛋白中, 部分可能是参与滞育相关的蛋白质网络中的成员, 它们可能在抗高温、 染色体分离、 叶酸代谢等生理过程中发挥重要作用。     

Proteomic analysis (GeLC-MS/MS) of ePFT-collected pancreatic fluid in chronic pancreatitis

Chronic pancreatitis is characterized by inflammation, fibrosis, pain, and loss of exocrine function of the pancreas. We aimed to identify differentially expressed proteins in the ePFT-collected pancreatic fluid from individuals with chronic pancreatitis (CP; n = 9) and controls with chronic abdominal pain not associated with the pancreas (NP; n = 9). Using GeLC-MS/MS techniques, we identified a total of 1391 different proteins in 18 pancreatic fluid samples. Of these proteins, 257 and 413 were identified exclusively in the control and chronic pancreatitis cohorts, respectively, and 721 were identified in both cohorts. Spectral counting and statistical analysis thereof revealed an additional 38 and 77 proteins that were up- or down-regulated, respectively, in the pancreatic fluid from individuals with chronic pancreatitis. As expected, gene ontology analysis illustrated that the largest percentage of differentially regulated proteins was secreted/extracellular in origin. In addition, proteins that were down-regulated with statistical significance in the chronic pancreatitis cohort were determined to have biological function of proteases, corresponding to the canonical pancreatic insufficiency associated with chronic pancreatitis. Proteins enriched in the pancreatic fluid of chronic pancreatitis patients had roles in fibrosis, inflammation, and pain, whereas digestive enzymes were significantly less abundant. Our workflow provided a mass spectrometry-based approach for the further study of the pancreatic fluid proteome, which may lead to the discovery potential biomarkers of chronic pancreatitis.     

Autoantibody against oxidized low-density lipoproteins may be enhanced by cigarette smoking

A total of 59 healthy male subjects (32 smokers and 27 nonsmokers) who had no reported systemic disease and did not take alcohol and vitamin supplementation were included. The levels of autoantibody to oxidized low-density lipoproteins (ox-LDL) in smokers and age-matched nonsmokers were compared. The plasma levels of antioxidants that can affect the formation of ox-LDL were also measured, and correlation analyses between anti ox-LDL IgG and plasma antioxidants, controlling for age and body mass index (BMI), were performed. Plasma alpha-tocopherol and uric acid concentrations of nonsmokers (2.78+/-1.09 microg/mg total lipid and 6.96+/-1.69 mg/dl, respectively) were significantly higher than those of smokers (1.68+/-0.48 microg/mg total lipid and 6.15+/-1.14 mg/dl, respectively) (P<0.05). Although plasma ascorbate and retinol levels were not significantly different between smokers and nonsmokers, smokers older than 45 years old had significantly lower plasma ascorbate levels (0.32+/-0.17 mg/dl) than age-matched nonsmokers (0. 53+/-0.14 mg/dl) (P=0.036). Higher level of plasma anti ox-LDL IgG was noted in the group of smokers compared with nonsmokers (515+/-409 mU/ml vs. 407+/-268 mU/ml, respectively) under the statistic method of Chi-Square test (P=0.049). A significant negative correlation was found between plasma anti ox-LDL IgG and alpha-tocopherol in the combined population as well as in the smoker group (r=-0.26, p=0.047; r=-0.48, p=0.006; respectively). However, there was no correlation between plasma anti ox-LDL IgG and the levels of other antioxidants. These results suggest that reduced concentrations of alpha-tocopherol are associated with cigarette smoking. The significantly negative correlation between plasma anti ox-LDL IgG and alpha-tocopherol in the entire study population as well as in the smoker group suggests that plasma alpha-tocopherol may be partially effective if not totally at protecting LDL from oxidative damage caused by cigarette smoking and dietary supplementation with alpha-tocopherol may provide a protective effect against LDL oxidation, especially in smokers.     

Smoke-induced signal molecules in bone marrow cells from altered low-density lipoprotein receptor-related protein 5 mice

Mechanism underlying smoke-induced loss of bone mass is unknown. In this study, we hypothesized that protein signals induced by smoking in bone marrow may be associated with the loss of bone mass. Using a proteomics approach, we identified 38 proteins differentially expressed in bone marrow cells from low-density lipoprotein receptor-related protein 5 (Lrp5) mice exposed to cigarette smoking. Smoking effects on protein expression in bone marrow among three genotypes (Lrp5(+/+), Lrp5(G171V), and Lrp5(-/-)) varied. On the basis of the ratio of protein expression induced by smoking versus nonsmoking, smoke induced protein expression significantly in wild-type mice compared to the other two genotypes (Lrp5(G171V) and Lrp5(-/-)). These proteins include inhibitors of β-catenin and proteins associated with differentiation of osteoclasts. We observed that S100A8 and S100A9 were overexpressed in human smokers compared to nonsmokers, which confirmed the effect of smoking on the expression of two proteins in Lrp5 mice, suggesting the role of these proteins in bone remodeling. Smoke induced expression of S100A8 and S100A9 in a time-dependent fashion, which was opposite of the changes in the ratio of OPG/RANKL in bone marrow cells, suggesting that the high levels of S100A8 and S100A9 may be associated with smoke-induced bone loss by increasing bone resorption.     

Proteomic analysis of an immortalized mouse pancreatic stellate cell line identifies differentially-expressed proteins in activated vs nonproliferating cell states

Pancreatic stellate cells (PaSC) are mediators in chronic pancreatitis and pancreatic cancer pathogenesis. Proteins regulating the biomolecular pathways involved in the conversion of activated to quiescent PaSC may have a significant influence in the development of chronic pancreatitis. We aim to compare differentially expressed proteins from an immortalized cell line of mouse PaSC in the activated and serum-starved cell states using mass spectrometry-based proteomics. PaSC cultured in media supplemented with fetal bovine serum (FBS) proliferate in the activated state, while serum starvation promotes the cellular transition to a "pseudo-quiescent" state. Using these two cell states, we performed a comparative mass spectrometry (GeLC-MS/MS) proteomic analysis. We identified over 2000 nonredundant proteins in PaSC. Qualitative and label-free quantitative analysis revealed several hundred proteins that were differentially abundant between the cell states. Proteins that were more abundant in activated PaSC included cytoskeletal proteins and ribosomal proteins, while those more abundant in pseudoquiescent PaSC included proteins involved in protein degradation-related pathways (lysosome, ubiquitin-mediated proteolysis, and the proteasome). Investigation of the role of PaSC in the pathogenesis of chronic pancreatitis using the mass spectrometry-based proteomics strategy described herein will lead to further insights into the molecular mechanisms associated with the disease.     

Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions

Streptococcus mutans, a major etiological agent of dental caries, causes demineralization of the tooth tissue due to the formation of acids from dietary carbohydrates. Dominant among the virulence determinants of this organism are aciduricity and acidogenicity, the abilities to grow at low pH and to produce acid, respectively. The mechanisms underlying the ability of S. mutans to survive and proliferate at low pH are currently under investigation. In this study we cultured S. mutans at pH 5.2 or 7.0 and extracted soluble cellular proteins. These were analyzed using high-resolution two-dimensional gel electrophoresis, and replicate maps of proteins expressed under each of the two conditions were generated. Proteins with modulated expression at low pH, as judged by a change in the relative integrated optical density, were excised and digested with trypsin by using an in-gel protocol. Tryptic digests were analyzed using matrix-assisted laser desorption ionization mass spectrometry to generate peptide mass fingerprints, and these were used to assign putative functions according to their homology with the translated sequences in the S. mutans genomic database. Thirty individual proteins exhibited altered expression as a result of culture of S. mutans at low pH. Up-regulated proteins (n = 18) included neutral endopeptidase, phosphoglucomutase, 60-kDa chaperonin, cell division proteins, enolase, lactate dehydrogenase, fructose bisphosphate aldolase, acetoin reductase, superoxide dismutase, and lactoylglutathione lyase. Proteins down-regulated at pH 5.2 (n = 12) included protein translation elongation factors G, Tu, and Ts, DnaK, small-subunit ribosomal protein S1P, large-subunit ribosomal protein L12P, and components of both phosphoenolpyruvate:protein phosphotransferase and multiple sugar binding transport systems. The identification of proteins differentially expressed following growth at low pH provides new information regarding the mechanisms of survival and has identified new target genes for mutagenesis studies to further assess their physiological significance.     

Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation

Respiratory diseases are an important health problem throughout the world. Whether caused by industrial pollutants, infections, smoking, cancer or metabolic diseases, damage to the lungs and airways often lead to morbidity or death. Bronchoalveolar lavage (BAL) obtained by fiber-optic bronchoscopy is a biofluid mirroring the expression of normally secreted pulmonary proteins and the products of activated cells and destructive processes. The characterization of the proteome within this compartment provides an opportunity to establish temporal and prognostic indicators of airway disease. The objective of this study was to develop methods of analysis of BAL samples, which achieved the highest level of annotation of the expression map of this proteome. We have optimized the process of sample preparation after investigating a variety of techniques including dialysis, ultramembrane filtration, precipitation and gel filtration. We have further studied methods to remove albumin from BAL in order to unmask proteins hidden on two-dimensional gels. In a pilot application of the method, BAL protein profiles obtained from healthy nonsmokers and smokers at risk for developing chronic obstructive pulmonary disease showed distinct differences.     

Sister chromatid exchanges in the peripheral blood of cigarette smokers and in lung cancer patients; and the effect of chemotherapy

Summary Peripheral blood sister chromatid exchange (SCE) rates in chronic cigarette smokers and in subjects with cancer do not differ from those in healthy nonsmokers. SCE patterns were normal in 69 chronic cigarette smokers, including 62 patients with untreated lung cancer. In three chronic smokers with lung cancer, high SCE levels were related to recent intravenous chemotherapy.Presented in part at the 15th Annual Somatic Cell Genetics Conference, Norfolk, Virginia, November 10–12, 1976 (Hollander et al., 1977)     

Enhanced protein glutathiolation and oxidative stress in cigarette smokers

There are many functional assays of oxidative damage to DNA, protein, and lipids but few reliable markers of chronic oxidative stress. The glutathiolation of proteins at key Cys residues is considered an important redox-sensitive, posttranslational signaling mechanism in the regulation of critical cellular functions. To determine whether protein bound glutathione (GSSP) is a sensitive indicator of oxidative stress, red blood cell and plasma concentrations were measured and compared between smokers and nonsmokers. In a community-based study conducted in Westchester County, New York, USA, blood samples were obtained from 354 cigarette smokers and 97 never smokers. The mean concentration of blood GSSP (micromol/L) was 32% higher in cigarette smokers and 43% higher when standardized by hemoglobin concentrations (p <.01). Plasma GSSP levels were also 20% higher in smokers than in nonsmokers (p <.001). The relationship was dose-dependent, with blood GSSP levels significantly correlated with cigarettes smoked per day, plasma cotinine, and plasma thiocyanate (r values ranged from .25 to .40). In smokers, there were no significant differences in GSSP and GSH levels by GSTM1 or GSTM3 genotype. Intraindividual variation in blood samples, as determined by taking serial samples over a 2-week period, was low (CV = 12.1%, n = 8). GSSP levels are stable over time but increase in response to the abundant free radicals in cigarette smoke. These findings support the use of GSSP as a sensitive biomarker of oxidative stress.     

Identification of differentially expressed proteins in blood plasma of control and cigarette smoke-exposed mice by 2-D DIGE/MS

Cigarette smoke exposure is known to induce obstructive lung disease and several cardiovascular disease states in humans and also in animal models. Smoking leads to oxidative stress and inflammation that are important in triggering pulmonary and cardiovascular disease. The objective of the current study was to quantify differences in expression levels of plasma proteins of cigarette smoke -exposed and control mice, at the time of disease onset, and identify these proteins for use as potential biomarkers of the onset of smoking-induced disease. We utilized 2-D DIGE/MS to characterize these proteomic changes. 2-D DIGE of plasma samples identified 11 differentially expressed proteins in cigarette smoke -exposed mice. From these 11 proteins, 9 were downregulated and 2 were upregulated. The proteins identified are involved in vascular function, coagulation, metabolism and immune function. Among these, the alterations in fibrinogen (2.2-fold decrease), α-1-antitrypsin (1.8-fold increase) and arginase (4.5-fold decrease) are of particular interest since these have been directly linked to cardiovascular and lung pathology. Differences in expression levels of these proteins were also confirmed by immunoblotting. Thus, we observe that chronic cigarette smoke exposure in mice leads to prominent changes in the protein expression profile of blood plasma and these changes in turn can potentially serve as markers predictive of the onset and progression of cardiovascular and pulmonary disease.     

Proteomic analysis of a rat pancreatic stellate cell line using liquid chromatography tandem mass spectrometry (LC-MS/MS)

Pancreatic stellate cells (PaSC) are emerging as key mediators in chronic pancreatitis and pancreatic cancer pathogenesis. Proteins regulating the biomolecular pathways involved in the conversion of quiescent to activated PaSC may have a significant influence on the development of chronic pancreatitis. We aim to compare differentially expressed proteins in activated and serum-starved non-proliferating PaSC using a mass spectrometry-based proteomics strategy. We cultured an immortalized rat PaSC cell line in media supplemented with 10% fetal bovine serum and in serum-free media. Using gel-based mass spectrometry (GeLC-MS/MS), we identified nearly 1500 proteins. Qualitative and quantitative proteomic analysis revealed several hundred proteins as differentially abundant between the two cell states. Proteins of greater abundance in activated PaSC included isoforms of actin (e.g., smooth muscle actin) and ribosomal proteins. Conversely, proteins more abundant in non-proliferating PaSC than in activated PaSC included signaling proteins MAP kinase 3 and Ras-related proteins. In addition, we have determined the molecular functions and biological pathways for these proteins. We are confident that the application of mass spectrometry-based strategies, such as that described herein, to investigate specific proteins in PaSC may lead to a better understanding of the molecular mechanisms involved in pancreatic diseases, such as chronic pancreatitis.     

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n?=?18) or slow (n?=?18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.     

Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.