Comparative Evaluation of Data Dependent and Data Independent Acquisition Workflows Implemented on an Orbitrap Fusion for Untargeted Metabolomics.

Constant improvements to the Orbitrap mass analyzer, such as acquisition speed, resolution, dynamic range and sensitivity have strengthened its value for the large-scale identification and quantification of metabolites in complex biological matrices. Here, we report the development and optimization of Data Dependent Acquisition (DDA) and Sequential Window Acquisition of all THeoretical fragment ions (SWATH-type) Data Independent Acquisition (DIA) workflows on a high-field Orbitrap FusionTM TribridTM instrument for the robust identification and quantification of metabolites in human plasma. By using a set of 47 exogenous and 72 endogenous molecules, we compared the efficiency and complementarity of both approaches. We exploited the versatility of this mass spectrometer to collect meaningful MS/MS spectra at both high- and low-mass resolution and various low-energy collision-induced dissociation conditions under optimized DDA conditions. We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.

Data acquisition workflows in liquid chromatography coupled to high resolution mass spectrometry-based metabolomics: Where do we stand?

Typical mass spectrometry (MS) based untargeted metabolomics protocols are tedious as well as time- and sample-consuming. In particular, they often rely on "full-scan-only" analyses using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) from which metabolites of interest are first highlighted, and then tentatively identified by using targeted MS/MS experiments. However, this situation is evolving with the emergence of integrated HRMS based-data acquisition protocols able to perform multi-event acquisitions. Most of these protocols, referring to as data dependent and data independent acquisition (DDA and DIA, respectively), have been initially developed for proteomic applications and have recently demonstrated their applicability to biomedical studies. In this context, the aim of this article is to take stock of the progress made in the field of DDA- and DIA-based protocols, and evaluate their ability to change conventional metabolomic and lipidomic data acquisition workflows, through a review of HRMS instrumentation, DDA and DIA workflows, and also associated informatics tools.

Technosphere insulin: defining the role of Technosphere particles at the cellular level.

BACKGROUND: Technosphere Insulin (TI) is a novel inhalation powder for the treatment of diabetes mellitus. Technosphere Insulin delivers insulin with an ultra rapid pharmacokinetic profile that is distinctly different from all other insulin products but similar to natural insulin release. Such rapid absorption is often associated with penetration enhancers that disrupt cellular integrity. METHODS: Technosphere Insulin was compared to a panel of known penetration enhancers in vitro using the Calu-3 lung cell line to investigate the effects of TI on insulin transport. RESULTS: Measures of tight junction integrity such as transepithelial electrical resistance, Lucifer yellow permeability, and F-actin staining patterns were all unaffected by TI. Cell viability and plasma membrane integrity were also not affected by TI. In contrast, cells treated with comparable (or lower) concentrations of penetration enhancers showed elevated Lucifer yellow permeability, disruption of the F-actin network, reduced cell viability, and compromised plasma membranes. CONCLUSIONS: These results demonstrate that TI is not cytotoxic in an in vitro human lung cell model and does not function as a penetration enhancer. Furthermore, TI does not appear to affect the transport of insulin across cellular barriers.