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.

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.

Optimizing Secondary Electrospray Ionization High-Resolution Mass Spectrometry (SESI-HRMS) for the Analysis of Volatile Fatty Acids from Gut Microbiome

Gut microbiota plays essential roles in maintaining gut homeostasis. The composition of gut microbes and their metabolites are altered in response to diet and remedial agents such as antibiotics. However, little is known about the effect of antibiotics on the gut microbiota and their volatile metabolites. In this study, we evaluated the impact of a moderate level of ampicillin treatment on volatile fatty acids (VFAs) of gut microbial cultures using an optimized real-time secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS). To evaluate the ionization efficiency, different types of electrospray solvents and concentrations of formic acid as an additive (0.01, 0.05, and 0.1%, v/v) were tested using VFAs standard mixture (C2–C7). As a result, the maximum SESI-HRMS signals of all studied m/z values were observed from water with 0.01% formic acid than those from the aqueous methanolic solutions. Optimal temperatures of sample inlet and ion chamber were set at 130 °C and 85 °C, respectively. SESI spray pressure at 0.5 bar generated the maximum intensity than other tested values. The optimized SESI-HRMS was then used for the analysis of VFAs in gut microbial cultures. We detected that the significantly elevated C4 and C7 VFAs in the headspace of gut microbial cultures six hours after ampicillin treatment (1 mg/L). In conclusion, our results suggested that the optimized SESI-HRMS method can be suitable for the analysis of VFAs from gut microbes in a rapid, sensitive, and non-invasive manner.

Minimizing ion competition boosts volatile metabolome coverage by secondary electrospray ionization orbitrap mass spectrometry

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HR-MS) is an emerging technique for the detection of volatile metabolites. However, sensitivity and reproducibility of SESI-HRMS have limited its applications in untargeted metabolomics profiling. Ion suppression in the SESI source has been considered to be the main cause. Here, we show that besides ion suppression, ion competition in the C-trap of Orbitrap instruments is another important factor that influences sensitivity and reproducibility of SESI-MS. Instead of acquiring the full mass-to-charge ratio (m/z) range, acquisition of consecutive m/z windows to minimize the ion competition effect allows the detection of more features. m/z window ranges are optimized to fill the C-trap either with an equal number of features or an equal cumulative intensity per window. Considering a balance between maximizing scanning speed and minimizing ion competition, splitting the m/z = 50-500 range into 4 windows is selected for measuring human breath and bacterial culture samples on SESI-Orbitrap MS, corresponding to a duty cycle of 2.3 s at a resolution of 140’000. In a small cohort of human subjects, the proposed splitting into 4 windows allows three times more features to be detected compared to the classical full m/z range method.