Profiling over 1500 lipids in induced lung sputum and the implications in studying lung diseases.

Induced lung sputum is a valuable matrix in the study of respiratory diseases. Although the methodology of sputum collection has evolved to a point where it is repeatable and responsive to inflammation, its use in molecular profiling studies is still limited. Here, an in-depth lipid profiling of induced lung sputum using high-resolution liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-Q-TOF MS) is described. An enormous complexity in lipid composition could be revealed. Over 1500 intact lipids, originating from 6 major lipid classes, have been accurately identified in 120 µL of induced sputum. By number and measured intensity, glycerophospholipids represent the largest lipid class, followed by sphingolipids, glycerolipids, fatty acyls, sterol lipids, and prenol lipids. Several prenol lipids, originating from tobacco, could be detected in the lung sputum of smokers. To illustrate the utility of the methodology in studying respiratory diseases, a comparative lipid screening was performed on lung sputum extracts in order to study the effect of Chronic Obstructive Pulmonary Disease (COPD) on the lung barrier lipidome. Results show that sphingolipid expression in induced sputum significantly differs between smokers with and without COPD.

Untargeted lipidomic analysis in chronic obstructive pulmonary disease. Uncovering sphingolipids.

RATIONALE: Cigarette smoke is the major risk factor in the development of chronic obstructive pulmonary disease (COPD). Lipidomics is a novel and emerging research field that may provide new insights in the origins of chronic inflammatory diseases, such as COPD. OBJECTIVES: To investigate whether expression of the sputum lipidome is affected by COPD or cigarette smoking. METHODS: Lipid expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry in induced sputum comparing smokers with and without COPD, and never-smokers. Changes in lipid expression after 2-month smoking cessation were investigated in smokers with and without COPD. MEASUREMENTS AND MAIN RESULTS: More than 1,500 lipid compounds were identified in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound level, 168 sphingolipids, 36 phosphatidylethanolamine lipids, and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared with smokers without COPD. The 13 lipids with a high fold change between smokers with and without COPD showed high correlations with lower lung function and inflammation in sputum. Twenty (glyco)sphingolipids and six tobacco-related compounds were higher expressed in smokers without COPD compared with never-smokers. Two-month smoking cessation reduced expression of 26 sphingolipids in smokers with and without COPD. CONCLUSIONS: Expression of lipids from the sphingolipid pathway is higher in smokers with COPD compared with smokers without COPD. Considering their potential biologic properties, they may play a role in the pathogenesis of COPD.

Critical role of aldehydes in cigarette smoke-induced acute airway inflammation.

BACKGROUND: Cigarette smoking (CS) is the most important risk factor for COPD, which is associated with neutrophilic airway inflammation. We hypothesize, that highly reactive aldehydes are critical for CS-induced neutrophilic airway inflammation. METHODS: BALB/c mice were exposed to CS, water filtered CS (WF-CS) or air for 5 days. Levels of total particulate matter (TPM) and aldehydes in CS and WF-CS were measured. Six hours after the last exposure, inflammatory cells and cytokine levels were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Furthermore, Beas-2b bronchial epithelial cells were exposed to CS extract (CSE) or WF-CS extract (WF-CSE) in the absence or presence of the aldehyde acrolein and IL-8 production was measured after 24 hrs. RESULTS: Compared to CS, in WF-CS strongly decreased (CS; 271.1 ± 41.5 µM, WF-CS; 58.5 ± 8.2 µM) levels of aldehydes were present whereas levels of TPM were only slightly reduced (CS; 20.78 ± 0.59 mg, WF-CS; 16.38 ± 0.36 mg). The numbers of mononuclear cells in BALF (p<0.01) and lung tissue (p<0.01) were significantly increased in the CS- and WF-CS-exposed mice compared to air control mice. Interestingly, the numbers of neutrophils (p<0.001) in BALF and neutrophils and eosinophils (p<0.05) in lung tissue were significantly increased in the CS-exposed but not in WF-CS-exposed mice as compared to air control mice. Levels of the neutrophil and eosinophil chemoattractants KC, MCP-1, MIP-1α and IL-5 were all significantly increased in lung tissue from CS-exposed mice compared to both WF-CS-exposed and air control mice. Interestingly, depletion of aldehydes in WF-CS extract significantly reduced IL-8 production in Beas-2b as compared to CSE, which could be restored by the aldehyde acrolein. CONCLUSION: Aldehydes present in CS play a critical role in inflammatory cytokine production and neutrophilic- but not mononuclear airway inflammation.

Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

LC-MS/MS quantification of next-generation biotherapeutics: a case study for an IgE binding Nanobody in cynomolgus monkey plasma.

BACKGROUND: Nanobodies(®) are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC-MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. RESULTS: Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC-MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. CONCLUSION: A 2D LC-MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.