Design and Implementation of a Dual-Probe Microsampling Apparatus for the Direct Analysis of Adherent Mammalian Cells by Ion Mobility-Mass Spectrometry

Abstract

Atmospheric pressure sampling mass spectrometric methods are ideal platforms for rapidly analyzing the metabolomes of biological specimens. Several liquid extraction-based techniques have been developed for increasing metabolome coverage in direct sampling workflows. Here, we report the construction of a dual-probe microsampling device (DPM), based on the design of the liquid microjunction surface sampling probe, for analyzing the metabolome of live microglial cells by drift-tube ion mobility spectrometry (IMS) quadrupole time-of-flight mass spectrometry. Utilizing two distinct solvent systems in parallel is demonstrated to extract a wide structural variety of metabolites and lipids, enabling a more comprehensive analysis of intracellular metabolism. Employing the DPM-IM-MS method to adherent cells yielded the detection of 73 unique lipids and 79 small molecule metabolites from each optimized solvent system probe, respectively. Integration of multiplexed ion mobility scans is also shown to increase extracted analyte signal intensities between 2- and 10-fold compared to traditional single-pulse IMS, enabling the detection of 38 low-intensity features not previously detected by single-pulse DPM-IM-MS. To examine the ability of the DPM system to differentiate between sample treatment groups, microglia were stimulated with the endotoxin lipopolysaccharide (LPS). Several metabolic alterations were detected between sample treatment groups by DPM-IM-MS, many of which were not previously detected with conventional single-probe liquid microjunction surface sampling.

Mass Spectrometry-Based Cellular Metabolomics: Current Approaches, Applications, and Future Directions

“…To further demonstrate the ability of LMJ-SS-MS to extract metabolites from mammalian cells, Comi et al have used optically guided MALDI-MS to profile the peptide content of single rat pancreatic islet cells followed by LMJ-SS extraction of the metabolites (primarily amino acids), analyzed offline by CE-MS. 178 This yielded a more comprehensive analysis of cellular metabolism in a semiautomated fashion, presenting an innovative technique for increasing metabolome coverage during a direct sampling approach. The construction of a dualprobe microsampling (DPM) system, based on the LMJ-SS design, has also been shown to increase metabolome coverage from live microglial cells by utilizing two distinct solvent systems in parallel to analyze a population of cells by IM-MS. 153 Each probe, composed of a distinct solvent system, enabled the simultaneous detection of structurally dissimilar molecules such as small molecule metabolites and lipids (Figure 4A). The use of the DPM apparatus further enabled the distinction between healthy and LPS-stimulated microglia, further demonstrating its novel analytical utility.…”

“…171 The spot size of LMJ-SS has been demonstrated in the range of 500−630 μm, limiting its use to the sampling of cell populations. 153,172 The majority of LMJ-SS-MS analyses have been implemented during the screening of microbial and fungal colonies for both genus identification and metabolome profiling. 173−176 The detection of secondary metabolites solely from a coculture system of two fungal strains was determined to be dependent on the interactions between the fungi, demonstrating the importance of coculture systems for better annotation of secreted metabolites, aiding in the field of natural product discovery.…”

“…The inner capillary draws in analyte rich solvent in preparation for ionization using pneumatic ESI, creating a liquid microjunction between the sample surface and probe tip, which allows for the continuous extraction of analytes . The spot size of LMJ-SS has been demonstrated in the range of 500–630 μm, limiting its use to the sampling of cell populations. , The majority of LMJ-SS-MS analyses have been implemented during the screening of microbial and fungal colonies for both genus identification and metabolome profiling. − The detection of secondary metabolites solely from a coculture system of two fungal strains was determined to be dependent on the interactions between the fungi, demonstrating the importance of coculture systems for better annotation of secreted metabolites, aiding in the field of natural product discovery . The use of this in situ microextraction technique enabled the mapping of fungal metabolites, helping to decipher the secretory mechanism of bioactive compounds from the fungi.…”

“…The integration of multiplexed drift-tube ion mobility scans prior to mass analysis has also been shown to aid in the detection of low-intensity analytes, assisting in the distinction between healthy and LPS-stimulated microglial cells by dual-probe microsampling. 153 Hence, the widespread use of IMS with traditional LC-MS and ambient sampling platforms could significantly aid in furthering the annotation of the metabolome of biological samples.…”

“…This experimental setup facilitated the detection of a wide range of metabolites and lipids originating from picoliter volumes of subcellular regions, further increasing the discriminatory power of TIMS-MS for structural identification using time aligned parallel fragmentation. The integration of multiplexed drift-tube ion mobility scans prior to mass analysis has also been shown to aid in the detection of low-intensity analytes, assisting in the distinction between healthy and LPS-stimulated microglial cells by dual-probe microsampling . Hence, the widespread use of IMS with traditional LC-MS and ambient sampling platforms could significantly aid in furthering the annotation of the metabolome of biological samples.…”