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Low molecular weight metabolites are essential for defining the molecular phenotypes of cells. However, spatial metabolomics tools often lack the sensitivity, specify, and spatial resolution to provide comprehensive descriptions of these species in tissue. MALDI imaging mass spectrometry (IMS) of low molecular weight ions is particularly challenging as MALDI matrix clusters are often nominally isobaric with multiple metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Additional difficulties surrounding low weight metabolite visualization include high resolution imaging, while maintaining sufficient ion numbers for broad and representative analysis of the tissue chemical complement. Here, we use MALDI timsTOF IMS to image low molecular weight metabolites at higher spatial resolution than most metabolite MALDI IMS experiments (20 µm) while maintaining broad coverage within the human kidney. We demonstrate that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different distributions within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for spatial metabolomics experiments. Through this improved sensitivity, we have found >40 low molecular weight metabolites in human kidney tissue such as arginic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. Future work will involve further exploring metabolomic profiles of human kidneys as a function of age, gender, and ethnicity.
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This content will become publicly available on April 29, 2026
Superior Differential Ion Mobility Spectrometry of Pendular Macromolecules Using Low-Frequency Rectangular Waveforms
Ion mobility spectrometry (IMS) can delineate gas-phase ions and probe their geometries. Coupling with electrospray ionization and MS has brought IMS to structural biology, revealing the macromolecular folding and subunit connectivity. However, the orientational averaging of ion–molecule collision cross sections (Ω) in the linear and field asymmetric waveform IMS (FAIMS) diminishes the resolution and structural specificity. In the novel low-field differential (LOD) IMS, a field too weak for ion heating (and thus FAIMS) aligns strong macrodipoles, capturing their magnitudes and directional Ω across the dipole (Ω⊥). However, the bisinusoidal waveforms (from FAIMS) have compromised the resolution, measurement accuracy, and correlation to the ion properties. Large ions amenable to LODIMS have low mobility and diffuse slowly, allowing the waveform frequencies down to ∼10 kHz. The low field and frequency permit generating the ideal rectangular waveforms with a flexible frequency and duty cycle by direct switching (impractical for FAIMS) in a miniature low-power format. This new IMS stage is evaluated for the exemplary large protein albumin (66 kDa) previously studied using the bisinusoidal waveform. The flat voltages and greater form factor initiate the differential IMS effect at lower fields, expand the separation space, and enable the quantification of Ω⊥ values by varying the duty cycle.
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- Award ID(s):
- 2105182
- PAR ID:
- 10616368
- Publisher / Repository:
- American Chemical Society (ACS)
- Date Published:
- Journal Name:
- Analytical Chemistry
- Volume:
- 97
- Issue:
- 16
- ISSN:
- 0003-2700
- Page Range / eLocation ID:
- 8841 - 8851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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