Differences in autophagy marker levels at birth in preterm vs. term infants.

BACKGROUND: Preterm infants are susceptible to oxidative stress and prone to respiratory diseases. Autophagy is an important defense mechanism against oxidative-stress-induced cell damage and involved in lung development and respiratory morbidity. We hypothesized that autophagy marker levels differ between preterm and term infants. METHODS: In the prospective Basel-Bern Infant Lung Development (BILD) birth cohort we compared cord blood levels of macroautophagy (Beclin-1, LC3B), selective autophagy (p62) and regulation of autophagy (SIRT1) in 64 preterm and 453 term infants. RESULTS: Beclin-1 and LC3B did not differ between preterm and term infants. However, p62 was higher (0.37, 95% confidence interval (CI) 0.05;0.69 in log2-transformed level, p = 0.025, padj = 0.050) and SIRT1 lower in preterm infants (-0.55, 95% CI -0.78;-0.31 in log2-transformed level, padj < 0.001). Furthermore, p62 decreased (padj-value for smoothing function was 0.018) and SIRT1 increased (0.10, 95% CI 0.07;0.13 in log2-transformed level, padj < 0.001) with increasing gestational age. CONCLUSION: Our findings suggest differential levels of key autophagy markers between preterm and term infants. This adds to the knowledge of the sparsely studied field of autophagy mechanisms in preterm infants and might be linked to impaired oxidative stress response, preterm birth, impaired lung development and higher susceptibility to respiratory morbidity in preterm infants. IMPACT: To the best of our knowledge, this is the first study to investigate autophagy marker levels between human preterm and term infants in a large population-based sample in cord blood plasma This study demonstrates differential levels of key autophagy markers in preterm compared to term infants and an association with gestational age This may be linked to impaired oxidative stress response or developmental aspects and provide bases for future studies investigating the association with respiratory morbidity.

UHPLC-MS/MS-Based Identity Confirmation of Amino Acids Involved in Response to and Side Effects from Antiseizure Medications.

Real-time breath analysis using secondary electrospray ionization coupled with high-resolution mass spectrometry is a fast and noninvasive method to access the metabolic state of a person. However, it lacks the ability to unequivocally assign mass spectral features to compounds due to the absence of chromatographic separation. This can be overcomed by using exhaled breath condensate and conventional liquid chromatography-mass spectrometry (LC-MS) systems. In this study, to the best of our knowledge, we confirm for the first time the presence of six amino acids (GABA, Oxo-Pro, Asp, Gln, Glu, and Tyr) previously reported to be involved in response to and side effects from antiseizure medications in exhaled breath condensate and by extension in exhaled human breath. Raw data are publicly available at MetaboLights with the accession number MTBLS6760.

Real-Time Volatile Metabolomics Analysis of Dendritic Cells.

Dendritic cells (DCs) actively sample and present antigen to cells of the adaptive immune system and are thus vital for successful immune control and memory formation. Immune cell metabolism and function are tightly interlinked, and a better understanding of this interaction offers potential to develop immunomodulatory strategies. However, current approaches for assessing the immune cell metabolome are often limited by end-point measurements, may involve laborious sample preparation, and may lack unbiased, temporal resolution of the metabolome. In this study, we present a novel setup coupled to a secondary electrospray ionization-high resolution mass spectrometric (SESI-HRMS) platform allowing headspace analysis of immature and activated DCs in real-time with minimal sample preparation and intervention, with high technical reproducibility and potential for automation. Distinct metabolic signatures of DCs treated with different supernatants (SNs) of bacterial cultures were detected during real-time analyses over 6 h compared to their respective controls (SN only). Furthermore, the technique allowed for the detection of 13C-incorporation into volatile metabolites, opening the possibility for real-time tracing of metabolic pathways in DCs. Moreover, differences in the metabolic profile of naïve and activated DCs were discovered, and pathway-enrichment analysis revealed three significantly altered pathways, including the TCA cycle, α-linolenic acid metabolism, and valine, leucine, and isoleucine degradation.

Increased Impact of Air Pollution on Lung Function in Preterm versus Term Infants: The BILD Study.

Rationale: Infants born prematurely have impaired capacity to deal with oxidative stress shortly after birth. Objectives: We hypothesize that the relative impact of exposure to air pollution on lung function is higher in preterm than in term infants. Methods: In the prospective BILD (Basel-Bern Infant Lung Development) birth cohort of 254 preterm and 517 term infants, we investigated associations of particulate matter ⩽10 µm in aerodynamic diameter (PM10) and nitrogen dioxide with lung function at 44 weeks' postconceptional age and exhaled markers of inflammation and oxidative stress response (fractional exhaled nitric oxide [FeNO]) in an explorative hypothesis-driven study design. Multilevel mixed-effects models were used and adjusted for known confounders. Measurements and Main Results: Significant associations of PM10 during the second trimester of pregnancy with lung function and FeNO were found in term and preterm infants. Importantly, we observed stronger positive associations in preterm infants (born 32-36 wk), with an increase of 184.9 (95% confidence interval [CI], 79.1-290.7) ml/min [Formula: see text]e per 10-µg/m3 increase in PM10, than in term infants (75.3; 95% CI, 19.7-130.8 ml/min) (pprematurity × PM10 interaction = 0.04, after multiple comparison adjustment padj = 0.09). Associations of PM10 and FeNO differed between moderate to late preterm (3.4; 95% CI, -0.1 to 6.8 ppb) and term (-0.3; 95% CI, -1.5 to 0.9 ppb) infants, and the interaction with prematurity was significant (pprematurity × PM10 interaction = 0.006, padj = 0.036). Conclusions: Preterm infants showed significantly higher susceptibility even to low to moderate prenatal air pollution exposure than term infants, leading to increased impairment of postnatal lung function. FeNO results further elucidate differences in inflammatory/oxidative stress response when comparing preterm infants with term infants.

Comparison of Plasma Ionization- and Secondary Electrospray Ionization- High-resolution Mass Spectrometry for Real-time Breath Analysis.

Real-time breath analysis by high-resolution mass spectrometry (HRMS) is a promising method to noninvasively retrieve relevant biochemical information. In this work, we conducted a head-to-head comparison of two ionization techniques: Secondary electrospray ionization (SESI) and plasma ionization (PI), for the analysis of exhaled breath. Two commercially available SESI and PI sources were coupled to the same HRMS device to analyze breath of two healthy individuals in a longitudinal study. We analyzed 58 breath specimens in both platforms, leading to 2,209 and 2,296 features detected by SESI-HRMS and by PI-HRMS, respectively. 60% of all the mass spectral features were detected in both platforms. However, remarkable differences were noted in terms of the signal-to-noise ratio (S/N), whereby the median (interquartile range, IQR) S/N ratio for SESI-HRMS was 115 (IQR = 408), whereas for PI-HRMS it was 5 (IQR = 5). Differences in the mass spectral profiles for the same samples make the inter-comparability of both techniques problematic. Overall, we conclude that both techniques are excellent for real-time breath analysis because of the very rich mass spectral fingerprints. However, further work is needed to fully understand the exact metabolic insights one can gather using each of these platforms.

Online Real-Time Monitoring of Exhaled Breath Particles Reveals Unnoticed Transport of Nonvolatile Drugs from Blood to Breath.

We used online secondary electrospray ionization mass spectrometry to measure venlafaxine (VEN), a nonvolatile drug, in the exhaled air of mice intraperitoneally treated with VEN. The breath pharmacokinetic (PK) profile of VEN was recorded, which was in good agreement with that of the blood. Combined with online collection of exhaled breath particles (EBPs), it was shown that VEN existed as part of EBPs rather than gas molecules in the breath. Linear free-energy relationship analysis confirmed that almost completely ionized VEN at physiological conditions unlikely partition from the lung lining fluid (LLF) into breath air. This implies that the occurrence of VEN in exhaled air accompanies the formation of EBPs from the LLF. By comparison with the low breath signals of VEN metabolites, passive membrane permeability and lung/blood partition coefficient are suggested as the main influencing factors for the levels of drugs in the breath. This study advances our knowledge on the mechanism by which nonvolatile drugs are transferred from blood into exhaled breath, providing guidance for breath test-based therapeutic drug monitoring.

Combination of Exhaled Breath Analysis with Parallel Lung Function and FeNO Measurements in Infants.

Breath analysis by secondary electrospray ionization-high resolution mass spectrometry (SESI-HRMS) offers the possibility to measure comprehensive metabolic profiles. The technology is currently being deployed in several clinical settings in Switzerland and China. However, patients are required to exhale directly into the device located in a dedicated room. Consequently, clinical implementation in patients incapable of performing necessary exhalation maneuvers (e.g., infants) or immobile (e.g., too weak, elderly, or in intensive care) remains a challenge. The aim of this study was to develop a method to extend such breath analysis capabilities to this subpopulation of patients by collecting breath samples remotely (offline) and promptly (within 10 min) transfer them to SESI-HRMS for chemical analysis. We initially assessed the method in adults by comparing breath mass spectra collected offline with Nalophan bags against spectra of breath samples collected in real time. In total, 13 adults provided 176 pairs of real-time and offline measurements. Lin's concordance correlation coefficient (CCC) was used to estimate the agreement between offline and real-time analyses. Here, 1249 mass spectral features (55% of total detected) exhibited Lin's CCC > 0.6. Subsequently, the method was successfully deployed to analyze breath samples from infants (n = 16), obtaining as a result SESI-HRMS breath profiles. To demonstrate the clinical feasibility of the method, we measured in parallel other clinical variables: (i) lung function, which characterizes the breathing patterns, and (ii) nitric oxide, which is a surrogate marker of airway inflammation. As a showcase, we focused our analysis on the exhaled oxidative stress marker 4-hydroxynonenal and its association with nitric oxide and minute ventilation.

Standardization procedures for real-time breath analysis by secondary electrospray ionization high-resolution mass spectrometry.

Despite the attractiveness of breath analysis as a non-invasive means to retrieve relevant metabolic information, its introduction into routine clinical practice remains a challenge. Among all the different analytical techniques available to interrogate exhaled breath, secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) offers a number of advantages (e.g., real-time, yet wide, metabolome coverage) that makes it ideal for untargeted and targeted studies. However, so far, SESI-HRMS has relied mostly on lab-built prototypes, making it difficult to standardize breath sampling and subsequent analysis, hence preventing further developments such as multi-center clinical studies. To address this issue, we present here a number of new developments. In particular, we have characterized a new SESI interface featuring real-time readout of critical exhalation parameters such as CO2, exhalation flow rate, and exhaled volume. Four healthy subjects provided breath specimens over a period of 1 month to characterize the stability of the SESI-HRMS system. A first assessment of the repeatability of the system using a gas standard revealed a coefficient of variation (CV) of 2.9%. Three classes of aldehydes, namely 4-hydroxy-2-alkenals, 2-alkenals and 4-hydroxy-2,6-alkedienals-hypothesized to be markers of oxidative stress-were chosen as representative metabolites of interest to evaluate the repeatability and reproducibility of this breath analysis analytical platform. Median and interquartile ranges (IQRs) of CVs for CO2, exhalation flow rate, and exhaled volume were 3.2% (1.5%), 3.1% (1.9%), and 5.0% (4.6%), respectively. Despite the high repeatability observed for these parameters, we observed a systematic decay in the signal during repeated measurements for the shorter fatty aldehydes, which eventually reached a steady state after three/four repeated exhalations. In contrast, longer fatty aldehydes showed a steady behavior, independent of the number of repeated exhalation maneuvers. We hypothesize that this highly molecule-specific and individual-independent behavior may be explained by the fact that shorter aldehydes (with higher estimated blood-to-air partition coefficients; approaching 100) mainly get exchanged in the airways of the respiratory system, whereas the longer aldehydes (with smaller estimated blood-to-air partition coefficients; approaching 10) are thought to exchange mostly in the alveoli. Exclusion of the first three exhalations from the analysis led to a median CV (IQR) of 6.7 % (5.5 %) for the said classes of aldehydes. We found that such intra-subject variability is in general much lower than inter-subject variability (median relative differences between subjects 48.2%), suggesting that the system is suitable to capture such differences. No batch effect due to sampling date was observed, overall suggesting that the intra-subject variability measured for these series of aldehydes was biological rather than technical. High correlations found among the series of aldehydes support this notion. Finally, recommendations for breath sampling and analysis for SESI-HRMS users are provided with the aim of harmonizing procedures and improving future inter-laboratory comparisons. Graphical abstract.

Molecular breath analysis supports altered amino acid metabolism in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Diagnosis of idiopathic pulmonary fibrosis (IPF) is complex and its pathogenesis is poorly understood. Recent findings indicate elevated levels of proline and other amino acids in lung tissue of IPF patients which may also be of diagnostic value. Following these findings, we hypothesized that such altered metabolic profiles would be mirrored in exhaled breath and could therefore be captured non-invasively in real time. METHODS: We aimed to validate these results using real-time exhaled breath analysis by secondary electrospray ionization-mass spectrometry, which can provide a non-invasive, painless and fast insight into the metabolism. Breath analysis was performed in a matched 1:1 case-control study involving 21 patients with IPF and 21 control subjects. RESULTS: We found significantly (P < 0.05) elevated levels of proline, 4-hydroxyproline, alanine, valine, leucine/isoleucine and allysine in breath of IPF patients, whereas pyroglutamic acid and phenylalanine did not show significant differences. This coincides with the amino acid's abundance in pulmonary tissue indicating that our observations reflect progressing fibrosis. In addition, amino acid levels correlated across subjects, further supporting a common underlying pathway. We were able to obtain a cross-validated area under the curve of 0.86, suggesting that these increased amino acid levels in exhaled breath have the potential to be used as biomarkers for IPF. CONCLUSION: We could validate previous findings of elevated lung tissue amino acid levels in IPF and show that online breath analysis might be a practical tool for a rapid screening for IPF.

Real-Time Monitoring of Tricarboxylic Acid Metabolites in Exhaled Breath.

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathway for cellular respiration in aerobic organisms. It provides and collects intermediates for many other interconnecting pathways and acts as a hub connecting metabolism of carbohydrates, fatty acids, and amino acids. Alteration in intracellular levels of its intermediates has been linked with a wide range of illnesses ranging from cancer to cellular necrosis or liver cirrhosis. Therefore, there exists an intrinsic interest in monitoring such metabolites. Our goal in this study was to evaluate whether, at least the most volatile metabolites of the TCA cycle, could be detected in breath in vivo and in real time. We used secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS) to conduct this targeted analysis. We enrolled six healthy individuals who provided full exhalations into the SESI-HRMS system at different times during 3 days. For the first time, we observed exhaled compounds that appertain to the TCA cycle: fumaric, succinic, malic, keto-glutaric, oxaloacetic, and aconitic acids. We found high intraindividual variability and a significant overall difference between morning and afternoon levels for malic acid, oxaloacetic acid, and aconitic acid, supporting previous studies suggesting circadian fluctuations of these metabolites in humans. This study provides first evidence that TCA cycle could conveniently be monitored in breath, opening new opportunities to study in vivo this important metabolic pathway.

Mass-Spectrometric Detection of Omega-Oxidation Products of Aliphatic Fatty Acids in Exhaled Breath.

Omega-oxidation is a fatty acid degradation pathway that can occur alternatively to the dominant ß-oxidation. The dysregulation of fatty acid oxidation has been related with a variety of diseases, termed fatty acid oxidation disorders. This work shows evidence for real-time detection in exhaled breath of the complete series of saturated linear ω-hydroxyalkanoic acids, ω-oxoalkanoic acids, and alkanedioic acids with carbon chain lengths of 5-15. We present a comprehensive analytical workflow using online and subsequent offline methods: secondary electrospray ionization mass spectrometry of exhaled breath and UHPLC-HRMS/MS experiments using exhaled breath condensate, respectively. By analyzing online breath measurements of 146 healthy individuals, we were able to obtain strong evidence for the correlation of these metabolite families. This enabled us to monitor the full ω-oxidation pathway in human exhaled breath. We could unambiguously identify these compounds, many of which have never been reported in breath so far. This comprehensive study on breath metabolites reinforces the notion of breath as a valuable source of information, which is underexploited in metabolomics.

Effects of CPAP therapy withdrawal on exhaled breath pattern in obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) is highly prevalent and associated with cardiovascular and metabolic changes. OSA is usually diagnosed by polysomnography which is time-consuming and provides little information on the patient's phenotype thus limiting a personalised treatment approach. Exhaled breath contains information on metabolism which can be analysed by mass spectrometry within minutes. The objective of this study was to identify a breath profile in OSA recurrence by use of secondary-electrospray-ionization-mass spectrometry (SESI-MS). METHODS: Patients with OSA effectively treated with CPAP were randomised to either withdraw treatment (subtherapeutic CPAP) or continue therapeutic CPAP for 2 weeks. Exhaled breath analysis by untargeted SESI-MS was performed at baseline and 2 weeks after randomisation. The primary outcome was the change in exhaled molecular breath pattern. RESULTS: 30 patients with OSA were randomised and 26 completed the trial according to the protocol. CPAP withdrawal led to a recurrence of OSA (mean difference in change of oxygen desaturation index between groups +30.3/h; 95% CI 19.8/h,40.7/h, p<0.001) which was accompanied by a significant change in 62 exhaled features (16 metabolites identified). The panel of discriminating mass-spectral features allowed differentiation between treated and untreated OSA with a sensitivity of 92.9% and a specificity of 84.6%. CONCLUSION: Exhaled breath analysis by SESI-MS allows rapid and accurate detection of OSA recurrence. The technique has the potential to characterise an individual's metabolic response to OSA and thus makes a comprehensible phenotyping of OSA possible. TRIAL REGISTRATION NUMBER: NCT02050425 (registered at ClinicalTrials.gov).

Capturing in Vivo Plant Metabolism by Real-Time Analysis of Low to High Molecular Weight Volatiles.

We have deployed an efficient secondary electrospray ionization source coupled to an Orbitrap mass analyzer (SESI-MS) to investigate the emissions of a Begonia semperflorens. We document how hundreds of species can be tracked with an unparalleled time resolution of 2 min during day-night cycles. To further illustrate the capabilities of this system for volatile organic compounds (VOCs) analysis, we subjected the plant to mechanical damage and monitored its response. As a result, ∼1200 VOCs were monitored displaying different kinetics. To validate the soundness of our in vivo measurements, we fully characterized some key compounds via tandem mass spectrometry (MS/MS) and confirmed their expected behavior based on prior gas chromatography/mass spectrometry (GC/MS) studies. For example, ß-caryophyllene, which is directly related to photosynthesis, was found to show a periodic day-night pattern with highest concentrations during the day. We conclude that the capability of SESI-MS to capture highly dynamic VOC emissions and wide analyte coverage makes it an attractive tool to complement GC/MS in plant studies.

Real-Time Quantification of Amino Acids in the Exhalome by Secondary Electrospray Ionization-Mass Spectrometry: A Proof-of-Principle Study.

BACKGROUND: Amino acids are frequently determined in clinical chemistry. However, current analysis methods are time-consuming, invasive, and suffer from artifacts during sampling, sample handling, and sample preparation. We hypothesized in this proof-of-principle study that plasma concentrations of amino acids can be estimated by measuring their concentrations in exhaled breath. A novel breath analysis technique described here allows such measurements to be carried out in real-time and noninvasively, which should facilitate efficient diagnostics and give insights into human physiology. METHODS: The amino acid profiles in 37 individuals were determined by ion-exchange HPLC in blood plasma and simultaneously in breath by secondary electrospray ionization coupled to high-resolution mass spectrometry. Participants were split into training and test sets to validate the analytical accuracy. Longitudinal profiles in 3 individuals were additionally obtained over a 12-h period. RESULTS: Concentrations of 8 slightly volatile amino acids (A, V, I, G, P, K, F, Orn) could be determined in exhaled breath with a CV of <10%. Exhalome validation studies yielded high accuracies for each of these amino acids, on average only 3% less compared to plasma concentrations (95% CI ±13%). Higher variations were found only for amino acids with a low plasma concentration. CONCLUSIONS: This study demonstrates for the first time that amino acids can be quantified in the human breath and that their concentrations correlate with plasma concentrations. Although this noninvasive technique needs further investigation, exhalome analysis may provide significant benefits over traditional, offline analytical methods.

Noninvasive strategies for breast cancer early detection.

Breast cancer screening and presurgical diagnosis are currently based on mammography, ultrasound and more sensitive imaging technologies; however, noninvasive biomarkers represent both a challenge and an opportunity for early detection of cancer. An extensive number of potential breast cancer biomarkers have been discovered by microarray hybridization or sequencing of circulating DNA, noncoding RNA and blood cell RNA; multiplex analysis of immune-related molecules and mass spectrometry-based approaches for high-throughput detection of protein, endogenous peptides, circulating and volatile metabolites. However, their medical relevance and their translation to clinics remain to be exploited. Once they will be fully validated, cancer biomarkers, used in combination with the current and emerging imaging technologies, represent an avenue to a personalized breast cancer diagnosis.

Identification of 2-alkenals, 4-hydroxy-2-alkenals, and 4-hydroxy-2,6-alkadienals in exhaled breath condensate by UHPLC-HRMS and in breath by real-time HRMS.

In recent years, breath analysis in real time has become a noninvasive alternative for the diagnosis of diseases and for molecular fingerprinting of exhaled breath. However, the techniques used lack the capabilities for proper identification of the compounds found in the exhalome. Here, we report the use of UHPLC-HRMS as a tool for the identification of several aldehydes (2-alkenals, 4-hydroxy-2-alkenals, and 4-hydroxy-2,6-alkadienals), biomarkers of lipid peroxidation, in exhaled breath condensate of three healthy subjects (N = 3). Some of the aldehydes studied have never been identified before. Their robust identification is based on retention times, on the generation of fragmentation trees from tandem mass spectra, and on the comparison of these parameters with standards. We also show that the identified compounds can be analyzed and confirmed by MS/MS in breath in real time and, therefore, they could be used as biomarkers for the rapid and noninvasive diagnosis of related diseases.

Drug Pharmacokinetics Determined by Real-Time Analysis of Mouse Breath.

Noninvasive, real-time pharmacokinetic (PK) monitoring of ketamine, propofol, and valproic acid, and their metabolites was achieved in mice, using secondary electrospray ionization and high-resolution mass spectrometry. The PK profile of a drug influences its efficacy and toxicity because it determines exposure time and levels. The antidepressant and anaesthetic ketamine (Ket) and four Ket metabolites were studied in detail and their PK was simultaneously determined following application of different sub-anaesthetic doses of Ket. Bioavailability after oral administration vs. intraperitoneal injection was also investigated. In contrast to conventional studies that require many animals to be sacrificed even for low-resolution PK curves, this novel approach yields real-time PK curves with a hitherto unmatched time resolution (10 s), and none of the animals has to be sacrificed. This thus represents a major step forward not only in animal welfare, but also major cost and time savings.

Breath analysis in real time by mass spectrometry in chronic obstructive pulmonary disease.

BACKGROUND: It has been suggested that exhaled breath contains relevant information on health status. OBJECTIVES: We hypothesized that a novel mass spectrometry (MS) technique to analyze breath in real time could be useful to differentiate breathprints from chronic obstructive pulmonary disease (COPD) patients and controls (smokers and nonsmokers). METHODS: We studied 61 participants including 25 COPD patients [Global Initiative for Obstructive Lung Disease (GOLD) stages I-IV], 25 nonsmoking controls and 11 smoking controls. We analyzed their breath by MS in real time. Raw mass spectra were then processed and statistically analyzed. RESULTS: A panel of discriminating mass-spectral features was identified for COPD (all stages; n = 25) versus healthy nonsmokers (n = 25), COPD (all stages; n = 25) versus healthy smokers (n = 11) and mild COPD (GOLD stages I/II; n = 13) versus severe COPD (GOLD stages III/IV; n = 12). A blind classification (i.e. leave-one-out cross validation) resulted in 96% sensitivity and 72.7% specificity (COPD vs. smoking controls), 88% sensitivity and 92% specificity (COPD vs. nonsmoking controls) and 92.3% sensitivity and 83.3% specificity (GOLD I/II vs. GOLD III/IV). Acetone and indole were identified as two of the discriminating exhaled molecules. CONCLUSIONS: We conclude that real-time MS may be a useful technique to analyze and characterize the metabolome of exhaled breath. The acquisition of breathprints in a rapid manner may be valuable to support COPD diagnosis and to gain insight into the disease.

Mass spectrometry research at the Laboratory for Organic Chemistry, ETH Zurich.

This contribution covers the most important activities of the Zenobi research group at the Organic Chemistry Laboratory, ETH Zurich. We work in a number of interrelated areas that encompass fundamental/mechanistic research, instrument and methods development, and applications. This is illustrated with examples from the mass spectrometric study of noncovalent interactions, using both native ESI and MALDI for ionization, the investigation of the gas-phase conformation of ionized bio-macromolecules, the use of ambient mass spectrometry for rapid, on-line analyses of, for example, exhaled breath, and the use of MALDI and microarray technologies for studying metabolites with extreme sensitivity, sufficient to probe the metabolites from single cells.