Secondary Electrospray Ionization Mass Spectrometry Research Articles

Abstract 4433: Secondary electrospray ionization mass spectrometry for volatile analysis of lung cancer

Abstract Lung cancer remains a leading cause of cancer-related mortality globally, necessitating innovative approaches for early detection and improved understanding of its metabolic landscape and consequences. Secondary Electrospray Ionization Mass Spectrometry (SESI-MS), an innovative analytical technique, has emerged as a powerful tool for the analysis of volatile organic compounds (VOCs) associated with various diseases, from metabolic disorders to bacterial infections. In this proof-of-concept study, we first utilized SESI-MS analyses of four representative lung cancer cells of both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) to demonstrate its capability to profile VOCs released by lung cancer cells. The technique allows for real-time, non-invasive analysis of the headspace above cultured cells, enabling the detection of unique VOC signatures associated with different lung cancer subtypes. Meanwhile, SESI-MS also facilitated the discrimination specific cells based on distinct VOC patterns. Furthermore, we also performed cisplatin treatment experiments and observed the metabolic signature shift post drug treatment. These results collectively offered potential applications in early diagnosis and personalized treatment strategies. Next, we continued with in vivo investigations to further extend the utility of SESI-MS in lung cancer research. Animal models, mimicking human lung cancer pathophysiology, have been subjected to SESI-MS analysis to identify volatile biomarkers indicative of tumor development and progression. Several representative cancer drug treatments were performed to the lung cancer mice model, and the drug-induced responses were also monitored via SESI-MS breath analyses. The in vivo SESI-MS approach detected many interesting breath VOC features that could provide valuable insights into the dynamic changes in VOC profiles during various stages of lung cancer, and therefore has the potential to offer a deeper understanding of the disease's metabolic alterations and potential avenues for targeted therapeutic interventions. In conclusion, SESI-MS-based volatile analysis presents a robust and versatile approach for investigating lung cancer, and our ongoing work has the potential to bridge the gap between in vitro and in vivo models for better translational research outcomes. The detection of unique VOC signatures associated with lung cancer cells and tumors holds significant promise for early detection, subtype classification, and monitoring of treatment responses. As technology continues to advance, SESI-MS stands poised to play a pivotal role in transforming the landscape of lung cancer diagnostics and research. Citation Format: Jiangjiang (Chris) Zhu, Fouad Choueiry. Secondary electrospray ionization mass spectrometry for volatile analysis of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4433.

Secondary-Electrospray Ionization Mass Spectrometry-Based Online Analyses of Mouse Volatilome Uncover Gut Microbiome-Dictated Metabolic Changes in the Host.

The symbiotic relationship between the gut microbial population is capable of regulating numerous aspects of host physiology, including metabolism. Bacteria can modulate the metabolic processes of the host by feeding on nutritional components within the lumen and releasing bioactive components into circulation. Endogenous volatile organic compound (VOC) synthesis is dependent on the availability of precursors found in mammalian metabolism. Herein, we report that microbial-mediated metabolic influences can alter the host volatilome and the prominent volatile changes can be uncovered by a novel volatile analysis technique named secondary electrospray ionization mass spectrometry. Mice were subjected to an antibiotic cocktail to deplete the microbiome and then inoculated with either single strain bacteria or fecal matter transplantation (FMT) to replete the microbial population in the gut. VOC sampling was achieved by using an advanced secondary electrospray ionization (SESI) source that directly mounted onto a Thermo Q-Exactive high-resolution mass spectrometer (HRMS). A principal component analysis summarizing the volatile profiles of the mice revealed independent clustering of each strain of the FMT-inoculated groups, suggesting unique volatile profiles. The Mummichog algorithm uncovered phenylalanine metabolism as a significantly altered metabolic profile in the volatilome of the microbiome-repleted mice. Our results indicated that the systemic metabolic changes incurred by the host are translated to unique volatile profiles correlated to the diversity of the microbial population colonized within the host. It is thus possible to take advantage of SESI-HRMS-based platforms for noninvasive screening of VOCs to determine the contribution of various microbial colonization within human gut that may impact host health.

Exhalomics as a noninvasive method for assessing rumen fermentation in dairy cows: Can exhaled-breath metabolomics replace rumen sampling?

Previously, we used secondary electrospray ionization-mass spectrometry (SESI-MS) to investigate the diurnal patterns and signal intensities of exhaled (EX) volatile fatty acids (VFA) of dairy cows. The current study aimed to validate the potential of an exhalomics approach for evaluating rumen fermentation. The experiment was conducted in a switchback design, with 3 periods of 9 d each, including 7 d for adaptation and 2 d for sampling. Four rumen-cannulated original Swiss Brown (Braunvieh) cows were randomly assigned to 1 of 2 diet sequences (ABA or BAB): (A) low starch (LS; 6.31% starch on a dry matter basis) and (B) high starch (HS; 16.2% starch on a dry matter basis). Feeding was once per day at 0830 h. Exhalome (with the GreenFeed System), and rumen samples were collected 8 times to represent every 3 h of a day, and EX-VFA and ruminal (RM)-VFA were analyzed using SESI-MS and HPLC, respectively. Furthermore, the VFA concentration in the gas phase (HR-VFA) was predicted based on RM-VFA and Henry's Law (HR) constants. No interactions were identified between the types of diets (HS vs. LS) and the measurement methods on daily average VFA profiles (RM vs. EX or HR vs. EX), suggesting a consistent performance among the methods. Additionally, when the 3-h interval VFA data from HS and LS diets were analyzed separately, no interactions were observed between methods and time of day, indicating that the relative daily pattern of VFA molar proportions was similar regardless of the VFA measurement method used. The results revealed that the levels of acetate sharply increased immediately after feeding, trailed by an increase in the acetate:propionate ratio and a steady increase for propionate (2 h after feeding the HS diet, 4 h for LS), and butyrate. This change was more pronounced for the HS diet than the LS diet. However, there was no overall diet effect on the VFA molar proportions, although the measurement methods affected the molar proportions. Furthermore, we observed a strong positive correlation between the levels of RM and EX acetate for both diets (HS: r = 0.84; LS: r = 0.85), RM and EX propionate (r = 0.74), and RM and EX acetate:propionate ratio (r = 0.80). Both EX-VFA and RM-VFA exhibited similar responses to feeding and dietary treatments, suggesting that EX-VFA could serve as a useful proxy for characterizing RM-VFA molar proportions to evaluate rumen fermentation. Similar relationships were observed between RM-VFA and HR-VFA. In conclusion, this study underscores the potential of exhalomics as a reliable approach for assessing rumen fermentation. Moving forward, research should further explore the depth of exhalomics in ruminant studies to provide a comprehensive insight into rumen fermentation metabolites, especially across diverse dietary conditions.

Direct mass spectrometry analysis of exhaled human breath in real-time.

The molecular composition of exhaled human breath can reflect various physiological and pathological conditions. Considerable progress has been achieved over the past decade in real-time analysis of exhaled human breath using direct mass spectrometry methods, including selected ion flow tube mass spectrometry, proton transfer reaction mass spectrometry, extractive electrospray ionization mass spectrometry, secondary electrospray ionization mass spectrometry, acetone-assisted negative photoionization mass spectrometry, atmospheric pressure photoionization mass spectrometry, and low-pressure photoionization mass spectrometry. Here, recent developments in direct mass spectrometry analysis of exhaled human breath are reviewed with regard to analytical performance (chemical sensitivity, selectivity, quantitative capabilities) and applications of the developed methods in disease diagnosis, targeted molecular detection, and real-time metabolic monitoring.

Non-invasive monitoring of microbiota and host metabolism using secondary electrospray ionization-mass spectrometry

The metabolic "handshake" between the microbiota and its mammalian host is a complex, dynamic process with major influences on health. Dissecting the interaction between microbial species and metabolites found in host tissues has been a challenge due to the requirement for invasive sampling. Here, we demonstrate that secondary electrospray ionization-mass spectrometry (SESI-MS) can be used to non-invasively monitor metabolic activity of the intestinal microbiome of a live, awake mouse. By comparing the headspace metabolome of individual gut bacterial culture with the "volatilome" (metabolites released to the atmosphere) of gnotobiotic mice, we demonstrate that the volatilome is characteristic of the dominant colonizing bacteria. Combining SESI-MS with feeding heavy-isotope-labeled microbiota-accessible sugars reveals the presence of microbial cross-feeding within the animal intestine. The microbiota is, therefore, a major contributor to the volatilome of a living animal, and it is possible to capture inter-species interaction within the gut microbiota using volatilome monitoring.

Trends in Direct Breath Analysis by Secondary Electrospray Ionization Mass Spectrometry for Clinical Applications

Exhaled breath can reveal insights about the metabolic state of the human body through the endo- and exogenous compounds it contains. The extent of detectable compounds, however, was revolutionized by the application of mass spectrometry. More specifically, secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) enables the detection of a broad range of breath-derived compounds simultaneously and with high sensitivity. Together with its rapid and non-invasive nature, direct breath analysis by SESI-HRMS raised particular interest for clinical applications. Over the past years, various clinical trials successfully demonstrated the technology´s capability for biomarker discovery in exhaled breath in adults and more recently in children. Current challenges lie within the potential translation of SESI-HRMS into clinical settings and the requirements therein, such as biomarker identification and validation, which became a focus of more recent studies.
  

Differentiation of Cystic Fibrosis-Related Pathogens by Volatile Organic Compound Analysis with Secondary Electrospray Ionization Mass Spectrometry.

Identifying and differentiating bacteria based on their emitted volatile organic compounds (VOCs) opens vast opportunities for rapid diagnostics. Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) is an ideal technique for VOC-biomarker discovery because of its speed, sensitivity towards polar molecules and compound characterization possibilities. Here, an in vitro SESI-HRMS workflow to find biomarkers for cystic fibrosis (CF)-related pathogens P. aeruginosa, S. pneumoniae, S. aureus, H. influenzae, E. coli and S. maltophilia is described. From 180 headspace samples, the six pathogens are distinguishable in the first three principal components and predictive analysis with a support vector machine algorithm using leave-one-out cross-validation exhibited perfect accuracy scores for the differentiation between the groups. Additionally, 94 distinctive features were found by recursive feature elimination and further characterized by SESI-MS/MS, which yielded 33 putatively identified biomarkers. In conclusion, the six pathogens can be distinguished in vitro based on their VOC profiles as well as the herein reported putative biomarkers. In the future, these putative biomarkers might be helpful for pathogen detection in vivo based on breath samples from patients with CF.

Sensitivity of secondary electrospray ionization mass spectrometry to a range of volatile organic compounds: Ligand switching ion chemistry and the influence of Zspray™ guiding electric fields.

Secondary electrospray ionization (SESI) is currently only semi-quantitative. In the Zspray™ arrangement of SESI-MS, the transfer of ions from near atmospheric pressure to a triple quadrupole is achieved by guiding electric fields that partially desolvate both reagent and analyte ions which must be understood. Also, to make SESI-MS more quantitative, the mechanisms and the kinetics of the reaction processes, especially ligand switching reactions of hydrated hydronium reagent ions, H3 O+ (H2 O)n , with volatile organic compound (VOC) molecules, need to be understood. A modified Zspray™ ESI ion source operating at sub-atmospheric pressure with analyte sample gas introduced via an inlet coaxial with the spray was used. Variation of the ion-guiding electric fields was used to reveal the degree of desolvation of both reagent and analyte ions. The instrument sensitivity was determined for several classes of VOCs by introducing bag samples of suitably varying concentrations as quantified on-line using selected ion flow tube MS. Electric field desolvation resulted in largely protonated VOCs, MH+ , and their monohydrates, MH+ H2 O, and for some VOCs proton-bound dimer ions, MH+ M, were formed. There was a highly linear response of the ion signal to the measured VOC sample concentration, which provided the instrument sensitivities, S, for 25 VOCs. The startling results show very wide variations in S from near 0 to 1 for hydrocarbons, and up to 100, on a relative scale, for polar compounds such as monoketones and unsaturated aldehydes. The complex ion chemistry occurring in the SESI ion source, largely involving gas-phase ligand switching, results in widely variable sensitivities for different classes of VOCs. The sensitivity is observed to depend on the dipole moment and proton affinity of the analyte VOC molecule, M, and to decrease with the observed fraction of MH+ H2 O, but other yet unrecognized factors must play a significant role.

Ligand Switching Ion Chemistry: An SIFDT Case Study of the Primary and Secondary Reactions of Protonated Acetic Acid Hydrates with Acetone.

A study was performed of the reactions of protonated acetic acid hydrates, CH3COOHH+(H2O)n, with acetone molecules, CH3COCH3, using a selected ion flow-drift tube (SIFDT). The rationale for this study is that hydrated protonated organic molecules are major product ions in secondary electrospray ionization mass spectrometry (SESI-MS) and ion mobility spectrometry (IMS). Yet the formation and reactivity of these hydrates are only poorly understood, and kinetics data are only sparse. The existing SIFDT instrument in our laboratory was upgraded to include an octupole ion guide and a separate drift tube by which hydrated protonated ions can be selectively injected into the drift tube reactor and their reactions with molecules studied under controlled conditions. This case study shows that, in these hydrated ion reactions with acetone molecules, the dominant reaction process is ligand switching producing mostly proton-bound dimer ions (CH3COCH3)H+(CH3COOH), with minor branching into (CH3COCH3)H+(H2O). This switching reaction was observed to proceed at the collisional rate, while other studied hydrated ions reacted more slowly. An attempt is made to understand the reaction mechanisms and the structures of the reaction intermediate ions at the molecular level. Secondary switching reactions of the asymmetric proton-bound dimer ions lead to a formation of strongly bound symmetrical dimers (CH3COCH3)2H+, the terminating ion in this ion chemistry. These results strongly suggest that, in SESI-MS and IMS, the presence of a polar compound, like acetone in exhaled breath, can suppress the analyte ions of low concentration compounds like acetic acid thus compromising their quantification.

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.

Insights on the Working Principles of Secondary Electrospray Ionization High-Resolution Mass Spectrometry for Quantitative Analysis of Aerosol Chemical Composition

Real-time mass spectrometry (MS) has attracted increasing interest in environmental analysis due to its advantages in high time resolution, minimization of sampling artifact, and avoidance of time-consuming sample pretreatment. Among real-time MS methods, secondary electrospray ionization MS (SESI-MS) is showing great promise for the detection of organic compounds in atmospheric particulate matter. In this study, we demonstrated the working principles of secondary nanoelectrospray ionization (Sec-nESI) for real-time measurement of laboratory-generated organic aerosols using l-tartaric acid (TA) as a model compound. Factors affecting the detection of TA particles using a homemade Sec-nESI source coupled with a high-resolution mass spectrometer are systematically investigated. Temperature of ion transport capillary (ITC) was found to be the key factor in determining the ion signal intensity, which shows an increase of intensity by a factor of 100 from ITC temperature of 100–300 °C and could be attributed to more efficient desolvation and ionization. The characteristic fragment ion at m/z 72.99 was selected for quantitative analysis of TA at normalized collision energy of 50%, the optimal value applied during MS/MS analysis. Detection limit of 0.14 µg/m3 and a linear range of 0.2–2.97 µg/m3 are achieved. Satisfactory correlations between ion signal intensity and particle surface area (R2 = 0.969) and mass concentration (R2 = 0.967) were obtained. Although an equally good correlation was observed between signal intensity and particle surface area, the good correlation between signal intensity and particle mass concentration indicates that high solubility of TA ensures efficient dissolution of TA in the primary ESI droplets for further ionization.

Reagent and analyte ion hydrates in secondary electrospray ionization mass spectrometry (SESI-MS), their equilibrium distributions and dehydration in an ion transfer capillary: Modelling and experiments.

Secondary electrospray ionization (SESI) in a water spray environment at atmospheric pressure involves the reactions of hydrated hydronium reagent ions, H3 O+ (H2 O)n , with trace analyte compounds in air samples. Understanding the formation and dehydration of reagent and analyte ions is the foundation for meaningful quantification of trace compounds by SESI-mass spectrometry (MS). A numerical model based on gas-phase ion thermochemistry is developed that describes equilibria in H3 O+ (H2 O)n reagent cluster ion distributions and ligand switching reactions with polar NH3 molecules leading to equilibrated hydrated ammonium ions NH4 + (H2 O)m . The model predictions are compared with experimental results obtained using a cylindrical SESI source coupled to an ion-trap mass spectrometer via a heated ion transfer capillary. Non-polar isoprene, C5 H8 , was used to further probe the nature of the reagent ions. Equilibrium distributions of H3 O+ (H2 O)n ions and their reactions with NH3 molecules have been characterized by the model in the near-atmospheric pressure SESI source. NH3 analyte molecules displace H2 O ligands from the H3 O+ (H2 O)n ions at the collisional rate forming NH4 + (H2 O)m ions, which travel through the heated ion transfer capillary losing H2 O molecules. The data for variable NH3 concentrations match the model predictions and the C5 H8 test substantiates the notion of dehydration in the heated capillary. Large cluster ions formed in the SESI region are dehydrated to H3 O+ (H2 O)1,2,3 and NH4 + (H2 O)1,2 while passing through the heated capillary, and considerable diffusion losses also occur. This phenomenon is also predicted for other polar analyte molecules, A, that can undergo similar switching reactions, thus forming AH+ and AH+ (H2 O)m analyte ions.

Volatile compounds released by Nalophan; implications for selected ion flow tube mass spectrometry and other chemical ionisation mass spectrometry analytical methods.

Three analytical techniques were used to analyse the VOCs released into the nitrogen used to expand Nalophan bags, viz. gas chromatography/mass spectrometry (GC/MS), secondary electrospray ionization mass spectrometry (SESI-MS) and selected ion flow tube mass spectrometry (SIFT-MS). The most significant VOCs were identified and quantified by SIFT-MS as a function of storage time, temperature and humidity. The consistent results obtained by these three analytical methods identify 1,2-ethanediol (ethylene glycol) and 2-methyl-1,3-dioxolane as the major VOCs released by the Nalophan. Their concentrations are enhanced by increasing the bag storage temperature and time, reaching 170 parts-per-billion by volume (ppbv) for ethylene glycol and 34 ppbv for 2-methyl-1,3-dioxolane in humid nitrogen (absolute humidity of 5%) contained in an 8-L Nalophan bag stored at 37°C for 160 min. Using H3 O+ reagent ions for SIFT-MS and SESI-MS analyses, the following analyte ions (m/z values) are affected by the Nalophan impurities: 45, 63, 81, 89 and 99, which can compromise analyses of acetaldehyde, ethylene glycol, monoterpenes, acetoin, butyric acid, hexanal and heptane.

Characterization of spoilage‐related volatile organic compounds in packaged leaf salads

AbstractA study has been made of the volatile organic compounds (VOCs) released into the contained headspace of packaged lamb's lettuce (Valerianella locusta) plants, cut at the roots, and of separated rocket (Eruca sativa) and baby spinach (Spinacia oleracea) leaves. Direct real‐time analyses of the headspace were carried out on each of five consecutive days using selected‐ion flow‐tube mass spectrometry (SIFT‐MS) and secondary electrospray ionization mass spectrometry (SESI‐MS), together with concomitant off‐line analyses using solid‐phase microextraction (SPME) and gas chromatography mass spectrometry (GC‐MS). It was observed that the concentrations of organosulphur compounds, especially dimethyl sulphide (DMS) and dimethyl disulphide (DMDS) were relatively low in the headspace of fresh samples, but increased exponentially over the 5 days for rocket, increased to a lesser extent for spinach, and increased to an even lesser extent for lamb's lettuce. Methanol was detected in the headspace of even fresh samples of lamb's lettuce, and its concentration increased for the subsequent 2–3 days before decreasing. This trend was mirrored in the isoprenoids analysed, which are characteristic of plant tissues. These results show that direct real‐time VOC analyses by SIFT‐MS can be usefully exploited to monitor fresh leaf salad spoilage, and that SESI‐MS spectra show patterns that are characteristic of salad type and storage time.

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

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. 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. 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. 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.

Headspace Gas Monitoring of Gut Microbiota Using Targeted and Globally Optimized Targeted Secondary Electrospray Ionization Mass Spectrometry.

This study aimed to develop and incorporate a secondary electrospray ionization (SESI) setup in combination with both targeted tandem mass spectrometry (MS/MS) and a hybrid metabolomics technique, globally optimized targeted mass spectrometry (GOT-MS), to sensitively detect volatile metabolites from the headspace of in vitro gut microbial culture in a human colonic model (HCM). Two SESI-tandem mass spectrometry panels with a comparable number of targeted metabolites/features (77 compounds in the targeted SESI-MS/MS panel and 75 features in the SESI-GOT-MS/MS panel) were established. The analytical performance of the SESI-GOT-MS/MS method, as well as its biological capability, were examined and compared with the targeted SESI-MS/MS method. As a result, the SESI-GOT-MS/MS method detected a similar number of metabolic features with good reproducibility (coefficient of variation <10%) compared to the targeted SESI-MS/MS method. Both methods showed a comparable ability to differentiate the gut microbial culture with or without the addition of green tea extract (GTE) to a HCM. The results from analysis of variance (ANOVA) showed that similar numbers of compounds from targeted SESI-MS/MS and metabolic features from SESI-GOT-MS/MS have significant differences when comparing samples collected from different HCM treatment stages. Partial least-squares discriminant analysis (PLS-DA) indicated that both methods could clearly differentiate the stages of GTE treatment. In summary, we demonstrated that SESI-MS/MS in combination with either targeted or GOT approaches can be a useful tool for monitoring gut microbial metabolism and their response to perturbations.

Differentiating Antibiotic-Resistant Staphylococcus aureus Using Secondary Electrospray Ionization Tandem Mass Spectrometry.

Secondary electrospray ionization mass spectrometry (SESI-MS) is an innovative metabolomics approach that primarily focuses on the gas-phase analyte detection. In this study, we developed a secondary electrospray ionization tandem mass spectrometry (SESI-MS/MS) method with a homemade SESI-MS front end, for sensitive, reproducible and selective detection of headspace volatile organic compounds (VOCs) emitted from bacterial culture. The optimized SESI-MS/MS was applied to examine the VOC metabolome of a pair of isogenic methicillin-susceptible and resistant Staphylococcus aureus (MSSA and MRSA) strains. From the headspace of bacterial culture, twelve organic acids, and eight amino acids with the mass range of 40-300 Da were specifically targeted with two selected reaction monitoring (SRM) transitions. Our results indicated that MSSA and MRSA strains can be clearly differentiated via partial least-squares discriminant analysis (PLS-DA) based on their headspace metabolic profiles. Furthermore, we studied the stress response of MSSA and MRSA to antibiotics treatment. Our result showed that MSSA and MRSA generated dramatically changed VOC metabolic profiles in response to ampicillin, which indicated that SESI-MS/MS could also be used for antibiotic treatment response monitoring in future studies. This study showed that SESI-MS/MS VOC analysis provides an additional approach to the bacterial metabolome detection complementary to traditional aqueous phase metabolite analysis. To the best of our knowledge, this was the first time that SESI-MS/MS was applied to investigate the bacterial metabolic perturbations caused by antibiotic treatment.

On-line Breath Metabolomics in Respiratory Diseases using Secondary Electrospray Ionization-Mass Spectrometry

Every second we are exhaling hundreds of endogenous and exogenous compounds that originate from blood and lung tissue. Obtaining metabolic information via exhaled breath analysis has been an emerging topic since the 1970s. Secondary electrospray ionization-mass spectrometry is a relatively new technique to detect these metabolites on-line in a highly sensitive and specific fashion. Using this technique, several respiratory diseases, including chronic obstructive pulmonary disease, obstructive sleep apnea, idiopathic pulmonary fibrosis, asthma, and lung cancer have been investigated over the past years. Several new potential biomarkers for these diseases were identified and new metabolic insights into their pathophysiology could be obtained.

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 β-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.

Rapid fingerprinting of grape volatile composition using secondary electrospray ionization orbitrap mass spectrometry: A preliminary study of grape ripening

We present a rapid and sensitive method based on secondary electrospray ionization mass spectrometry (SESI-MS) for profiling volatile emissions from the intact berries of non-Muscat grape cultivars (Pinot Noir, Chardonnay and Sauvignon Blanc). The method does not require sample preparation or concentration steps. Grape volatiles were tentatively identified based on accurate mass, the related elemental composition and literature. Approximately 300 peaks were detected in positive ion mode, and fewer (70–100) in negative ion mode. We monitored changes in grape berry volatile composition during ripening to screen for potential ripeness markers and observed ten [M+H]+ peaks and two [M-H]- peaks that evolved in a significant linear trend (R2 ≥ 0.80, p < 0.05) for the combined data across all cultivars either increasing or decreasing in the final four weeks of ripening. Peaks assigned to C13-norisoprenoids and benzenoid derivatives have shown similar trends in previous studies using offline gas chromatography (GC) approaches. Principal components analysis showed that negative ion mode clearly separated each stage of grape ripeness, whilst positive ion mode only separated berries in the final stage, pre-harvest. From this preliminary study, we conclude that SESI-MS holds promise as a tool for rapid screening of grape volatiles. Some marker ions had no interfering peaks within a 1-Da window, such that they could be monitored with simple unit-resolution instruments in future studies. This implies that SESI-MS in combination with portable MS instrumentation has potential for field analysis where real-time analysis is key.