Resolving isobaric interferences in direct infusion tandem mass spectrometry.

RATIONALE: The co-fragmentation of precursors in direct infusion (DI) tandem high-resolution mass spectrometry (HRMS) can complicate the fragment spectra and consequently lead to false hits during compound identification. METHODS: The method herein described, termed IQAROS (incremental quadrupole acquisition to resolve overlapping spectra), modulates the intensities of precursors and fragments by stepwise movement of the quadrupole isolation window over the mass-to-charge (m/z) range of the precursors. The modulated signals are then deconvoluted by a linear regression model to reconstruct the fragment spectra with less interference. The hardware to demonstrate the use of IQAROS was an orbitrap with electrospray ionization (ESI) or secondary electrospray ionization (SESI), although the method can also be applied to other ionization techniques or mass analyzers. RESULTS: Assessing the performance of IQAROS with isobaric standards revealed that the reconstructed fragment spectra match with spectra acquired from the pure standards and that more compounds were correctly identified compared with the classical approach with the quadrupole centered at the m/z value of the precursor of interest. Moreover, the strength of IQAROS is exemplified by the identification of two isobaric biomarkers directly from a breath sample with SESI-HRMS. CONCLUSIONS: With IQAROS, cleaner fragment spectra of co-fragmenting isobars during DI-HRMS analysis can be obtained. IQAROS can easily be set up by the standard graphical user interface of the instrument. Therefore, it facilitates the characterization of features of interest in samples analyzed by DI-HRMS, for example, in high-throughput or real-time metabolomics.

Effects of a Volatile Organic Compound Filter on Breath Profiles Measured by Secondary Electrospray High-Resolution Mass Spectrometry.

Environmental volatile organic compounds (VOCs) from the ambient air potentially influence on-line breath analysis measurements by secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS). The aim of this study was to investigate how inhaling through a VOC filter affects the detected breath profiles and whether it is feasible to integrate such filters into routine measurements. A total of 24 adult participants performed paired breath analysis measurements with and without the use of an activated carbon filter for inspiration. Concordance correlation coefficients (CCCs) and the Bland−Altman analysis were used to assess the agreement between the two methods. Additionally, the effect on a selection of known metabolites and contaminants was analyzed. Out of all the detected features, 78.3% showed at least a moderate agreement before and after filter usage (CCC > 0.9). The decrease in agreement of the remaining m/z features was mostly associated with reduced signal intensities after filter usage. Although a moderate-to-substantial concordance was found for almost 80% of the m/z features, the filter still had an effect by decreasing signal intensities, not only for contaminants, but also for some of the studied metabolites. Operationally, the use of the filter complicated and slowed down the conductance of measurements, limiting its applicability in clinical studies.

Bioaffinity Screening with a Rapid and Sample-Efficient Autosampler for Native Electrospray Ionization Mass Spectrometry.

Fast and efficient handling of ligands and biological targets are required in bioaffinity screening based on native electrospray ionization mass spectrometry (ESI-MS). We use a prototype microfluidic autosampler, called the "gap sampler", to sequentially mix and electrospray individual small molecule ligands together with a target protein and compare the screening results with data from thermal shift assay and surface plasmon resonance. In a first round, all three techniques were used for a screening of 110 ligands against bovine carbonic anhydrase II, which resulted in five mutual hits and some false positives with ESI-MS presumably due to the high ligand concentration or interferences from dimethyl sulfoxide. In a second round, 33 compounds were screened in lower concentrations and in a less complex matrix, resulting in only true positives with ESI-MS. Within a cycle time of 30 s, dissociation constants were determined within an order of magnitude accuracy consuming only 5 pmol of ligand and less than 15 pmol of protein per screened compound. In a third round, dissociation constants of five compounds were accurately determined in a titration experiment. Thus, the gap sampler can rapidly and efficiently be used for high-throughput screening.

The Gap Sampler: A Versatile Microfluidic Autosampler for Electrospray Ionization Mass Spectrometry.

Microfluidic autosamplers for electrospray ionization mass spectrometry (ESI-MS) are of major importance when using ESI-MS as a high-throughput and low sample consumption analytical method. In this article, microfluidic ESI-MS autosampler designs are overviewed and a group-owned prototype is discussed. The socalled gap sampler is a pin-based sampler for miniaturized flow injection (FI) analysis. To date, it has been used in various applications. Following proof of concept applications with FI of small molecules, pin modifications were implemented for unspecific and specific extraction for the analysis of complex samples. Most recently, further optimization allowed the study of non-covalent protein-ligand interactions for bioaffinity screenings, which constitutes a major milestone in the development of this novel high-throughput autosampler.

Plasmon-Driven Photocatalysis Leads to Products Known from E-beam and X-ray-Induced Surface Chemistry.

Plasmonic metal nanostructures can concentrate incident optical fields in nanometer-sized volumes, called hot spots. This leads to enhanced optical responses of molecules in such a hot spot but also to chemical transformations, driven by plasmon-induced hot carriers. Here, we employ tip-enhanced Raman spectroscopy (TERS) to study the mechanism of these reactions in situ at the level of a single hot spot. Direct spectroscopic measurements reveal the energy distribution of hot electrons, as well as the temperature changes due to plasmonic heating. Therefore, charge-driven reactions can be distinguished from thermal reaction pathways. The products of the hot-carrier-driven reactions are strikingly similar to the ones known from X-ray or e-beam-induced surface chemistry despite the >100-fold energy difference between visible and X-ray photons. Understanding the analogies between those two scenarios implies new strategies for rational design of plasmonic photocatalytic reactions and for the elimination of photoinduced damage in plasmon-enhanced spectroscopy.

How Soft Is Secondary Electrospray Ionization?

Secondary electrospray ionization (SESI) mass spectrometry (MS) is a direct infusion technique often used for untargeted metabolomics, e.g., for online breath analysis. SESI is thought to be a soft ionization method, which is important to avoid interference from in-source fragments and to simplify compound annotation. In this work, benzylammonium ions, formed from volatile benzylamines, with known bond dissociation enthalpies were used as thermometer ions to investigate the internal energy distribution of ions that are produced by SESI. It is shown that SESI is softer than electrospray ionization (ESI), and therefore, SESI indeed qualifies as a soft ionization technique. However, we also found that the standard MS instrument settings used in the SESI community are relatively harsh. Proper soft tuning of the instrument is essential to fully benefit from the softness that SESI can provide. Moreover, there is evidence from in-source collision-induced dissociation (CID) experiments that analytes can be solvated in SESI under soft conditions, which supports a recently proposed SESI mechanism referred to as ligand switching.

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

Secondary electrospray ionization high-resolution mass spectrometry (SESI-HRMS) 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.

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.

Studying biomolecular folding and binding using temperature-jump mass spectrometry.

Characterizing folding and complex formation of biomolecules provides a view into their thermodynamics, kinetics and folding pathways. Deciphering kinetic intermediates is particularly important because they can often be targeted by drugs. The key advantage of native mass spectrometry over conventional methods that monitor a single observable is its ability to identify and quantify coexisting species. Here, we show the design of a temperature-jump electrospray source for mass spectrometry that allows one to perform fast kinetics experiments (0.16-32 s) at different temperatures (10-90 °C). The setup allows recording of both folding and unfolding kinetics by using temperature jumps from high to low, and low to high, temperatures. Six biological systems, ranging from peptides to proteins to DNA complexes, exemplify the use of this device. Using temperature-dependent experiments, the folding and unfolding of a DNA triplex are studied, providing detailed information on its thermodynamics and kinetics.