Integrating ion mobility spectrometry into MS-based exposome measurements Perspective Executive summary
Background • Human disease is a combination of individual genetic factors and nongenetic environmental factors. • The ‘exposome’ complements the genome and is the sum of all nongenetic exposures over a lifetime to chemical, social and biological agents.
• Omics technologies can assist in characterizing the exposome by directly measuring chemical exposures and by inferring exposure based on biological signatures obtained by one or more complementary approaches.
• As new analytical methodologies for measuring small molecules advance, there will be a need for computational approaches to rapidly and comprehensively identify chemicals and metabolites.
State-of-the-art in measuring the exposome • The technical approaches used to measure the exposome largely fall under one of two categories: targeted or untargeted. • Targeted analyses focus on a limited number of analytes, have high quantitative accuracy and low limits of quantification, but only measures a narrow snapshot of the sample molecular composition.
• Untargeted analyses do not focus on a specific analyte but instead seek to comprehensively measure all analytes in a sample and offer the best opportunity to discover novel markers of exposure. Caveats include possible artifacts in the data due to the lack of optimization of sample preparation procedures, difficulty detecting very low-abundance analytes in the presence of high- abundance analytes and an incomplete representation of chemical space in spectral reference libraries.
• NMR spectroscopy and LC-MS coupled with MS are typically employed in untargeted analysis of chemicals and metabolites. • Communicating the confidence in chemical identification can be a challenge and several systems exist for small molecules, including the Metabolomics Standards Initiative, an LC-high resolution MS/MS specific set from Eawag and many more. The essences of these are:
• Confirmed identification with two orthogonal matching properties to an authentic reference standard measured in-house (MSI Level 1, Eawag Level 1).
• Probable identification with all evidence indicating only one structure is possible, but authentic standard is not available for confirmation (Eawag Level 2a/b).
• Putative annotation based on physicochemical properties and spectral matching (MSI Level 2, Eawag Level 2a). • Tentative identification/Putative compound class – tentative identification using predictive techniques, multiple structures are possible or insufficient evidence to eliminate other structures; substance class only is clear (MSI Level 3, Eawag Level 3).
• Unknown compounds – molecular formula is unequivocal (Eawag Level 4) or exact mass only (Eawag Level 5; both MSI Level 4). These can be traced in samples and correspond to ‘detected features’ in the analyses, but the identity remains unclear.
Introducing ion mobility spectrometry as a new tool for the exposomics toolbox • Drift tube ion mobility spectrometry (DTIMS) shows great promise in small molecule measurements because it is able to directly determine molecular structural information.
• The ability to resolve isomers that are difficult to distinguish using LC-MS alone is an inherent strength of DTIMS, particularly in small molecule analysis.
• Because DTIMS instruments depend only on drift cell pressure, temperature and length, molecular collisional cross section (CCS) measurements are extremely reproducible. Furthermore, measurements from different instruments in different laboratories have also been compared and their values normally agree within <2% error, with recent DTIMS instruments yielding values with reproducibility precision of <1%.
• The high reproducibility and speed of DTIMS allows it to be easily nested between LC and MS to provide additional separation power and dynamic range of detection in measuring the exposome.
• When coupled with TOF time-of-flight MS, DTIMS-MS analysis can be ultra-high throughput, with a single ion mobility spectrometry (IMS) separation typically occurring in 10–100 ms.
• A novel, automated SPE sample introduction system coupled with DTIMS-MS provides a 10-s sample-to-sample duty cycle and a theoretical maximum throughput of >8000 injections per day.
• Accurate, computationally predicted CCS values can facilitate the broad identification of detected molecules in combination with accurate mass and MS/MS spectra, when available, and possibly with just accurate mass.
Future perspective • Analytical and data processing challenges remain for full-scale implementation of IMS in standard exposomics workflows. • Ultra-high resolution IMS devices constructed with Structures for Lossless Ion Manipulations are pushing the separation resolution possibilities of IMS.
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