Assessing the Potential of Untargeted SWATH Mass Spectrometry-Based Metabolomics to Differentiate Closely Related Exposures in Observational Studies.

Mass spectrometry (MS) is increasingly used in clinical studies to obtain molecular evidence of chemical exposures, such as tobacco smoke, alcohol, and drugs. This evidence can help verify clinical data retrieved through anamnesis or questionnaires and may provide insights into unreported exposures, for example those classified as the same despite small but possibly relevant chemical differences or due to contaminants in reported exposure compounds. Here, we aimed to explore the potential of untargeted SWATH metabolomics to differentiate such closely related exposures. This data-independent acquisition MS-based profiling technique was applied to urine samples of 316 liver and 570 kidney transplant recipients from the TransplantLines Biobank and Cohort Study (NCT03272841), where we focused on the immunosuppressive drug mycophenolate, which is either supplied as a morpholino-ester prodrug or as an enteric-coated product, the illicit drug cocaine, which is usually supplied as an adulterated product, and the proton pump inhibitors omeprazole and esomeprazole. Based on these examples, we found that untargeted SWATH metabolomics has considerable potential to identify different (unreported) exposure or co-exposure metabolites and may determine variations in their abundances. We also found that these signals alone may sometimes be unable to distinguish closely related exposures, and enhancement of differentiation, for example by integration with pharmacogenomics data, is needed.

SWATH data independent acquisition mass spectrometry for screening of xenobiotics in biological fluids: Opportunities and challenges for data processing.

SWATH data independent acquisition (DIA) mass spectrometry (MS) has become an established technique in MS-based 'omics' research and is increasingly used for the screening of xenobiotics (e.g. drugs, drug metabolites, pesticides, toxicants). Such xenobiotic screening methods are mostly applied for tentative compound identification purposes based on spectral library searching, while additional data processing techniques are scarcely used thereby leaving the full potential of these methods often unused. Here we present an analytical workflow for screening xenobiotics in human samples using SWATH/MS based on which we highlight opportunities for unlocking unused potential of these methods. The workflow was applied to urine samples from subjects who tested positive for THC and/or cocaine during roadside drug testing with the goal of confirming the positive roadside drug tests and identifying compounds that relate to illicit drug use (e.g. cutting agents, tobacco components) or associate with corresponding lifestyle choices (e.g. nasal decongestants, painkillers). These goals could only be reached by complementing spectral library search procedures with additional multivariate data analyses due to inherent incompleteness of the spectral library that was employed. Such incompleteness represents a common challenge for applications where limited or no metadata is available for study samples, for example in toxicology, doping control in sports, and workplace or roadside drug testing. It furthermore sets the stage for employing additional data processing techniques as is outlined in the presented work.

Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

Methodological considerations in the development of HPLC-MS methods for the analysis of rodent plasma for metabonomic studies.

A study of the factors involved in obtaining valid global metabolite profiles from the HPLC-MS of rat or mouse plasma for the purposes of metabonomic analysis has been undertaken. Plasma proteins were precipitated with three volumes of either methanol or acetonitrile. Chromatographic separations were performed on a C18-bonded stationary phase using 3.5 and 5 mum particles packed into 2.1 and 4.6 mm i.d. formats, respectively, and on a C8 phase using 3.5 mum particles and a 2.1 mm i.d. column. Three reversed-phase gradient solvent systems, based on acidified water-acetonitrile, acidified water-methanol and acidified water-methanol-acetonitrile mixtures, were investigated. The column eluent was analysed with both positive and negative electrospray ionisation using a quadrupole-linear ion trap mass spectrometer. These studies revealed that while accurate classification of sample type can be made, there are a number of methodological problems associated with the analysis of plasma with respect to factors such as repeatability and column longevity. In particular, special care has to be taken to ensure that the analytical system is properly "conditioned" by the repeated injection of matrix samples. The use of biological quality control (QC) samples provided an important means of monitoring method performance. Finally, the source of the plasma (Zucker wild-type or (fa/fa) rat or mouse tumour model) also appeared to have an effect on the repeatability of the methodology.