Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform‐specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano‐DESI MSI) for the proteoform‐selective imaging of biological tissues. Nano‐DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof‐of‐concept experiments demonstrate that nano‐DESI MSI combined with on‐tissue top‐down proteomics is ideally suited for the proteoform‐selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.
Nanospray Desorption Electrospray Ionization (nano-DESI) Mass Spectrometry Imaging of Drift Time-Separated Ions
Simultaneous spatial localization and structural characterization of molecules in complex biological samples
currently represents an analytical challenge for mass spectrometry imaging (MSI) techniques. In this study,
we describe a novel experimental platform, which substantially expands the capabilities and enhances the
depth of chemical information obtained in high spatial resolution MSI experiments performed using nanospray
desorption electrospray ionization (nano-DESI). Specifically, we designed and constructed a portable
nano-DESI MSI platform and coupled it with a drift tube ion mobility spectrometer-mass spectrometer
(IM-MS). Separation of biomolecules observed in MSI experiments based on their drift times provides unique
molecular descriptors necessary for their identification by comparison with databases. Furthermore, it enables
isomer-specific imaging, which is particularly important for unraveling the complexity of biological systems.
Imaging of day 4 pregnant mouse uterine sections using the newly developed nano-DESI-IM-MSI system demonstrates rapid isobaric and isomeric separation and reduced chemical noise in MSI experiments. A direct comparison of the performance of the new nano-DESI-MSI platform operated in the MS mode with the more established nano-DESI-Orbitrap platform indicates a comparable performance of these two systems. A spatial resolution of better than ~16 μm and similar molecular coverage was obtained using both platforms. The structural information provided by the ion mobility separation expands the molecular specificity of high-resolution MSI necessary for the detailed understanding of biological systems.
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- Award ID(s):
- 1808136
- PAR ID:
- 10210472
- Date Published:
- Journal Name:
- ChemRxiv
- ISSN:
- 2573-2293
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform‐specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano‐DESI MSI) for the proteoform‐selective imaging of biological tissues. Nano‐DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof‐of‐concept experiments demonstrate that nano‐DESI MSI combined with on‐tissue top‐down proteomics is ideally suited for the proteoform‐selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.
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null (Ed.)Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns of toxicity as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, little is known about their localization in tissues. Herein, we used nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI-MSI) to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. Nano-DESI allows for label-free imaging with high spatial resolution and sensitivity without special sample pretreatment. Using nano-DESI-MSI, ion images for diclofenac and its major metabolites were produced. MSI data acquired over a broad m/z range showed fairly low signals of the drug and its metabolites. At least an order of magnitude improvement in the signals was obtained using selected ion monitoring (SIM), with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide is localized to the inner medulla and hydroxydiclofenac to the cortex of the kidney. The distributions observed for both metabolites closely match the previously reported localization of enzymes that process diclofenac into its respective metabolites. The localization of diclofenac acyl glucuronide to medulla likely indicates that the toxic metabolite is being excreted from the tissue. In contrast, a uniform distribution of diclofenac, hydroxydiclofenac and the diclofenac acyl glucuronide metabolite was observed in the liver tissue. Semiquantitative analysis found the metabolite to diclofenac ratios calculated from nano-DESI in agreement to those calculated from liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments. Collectively, our results demonstrate nano-DESI-MSI can be successfully used to image diclofenac and its primary metabolites in dosed liver and kidney tissues from mice and derive semi-quantitative data from localized tissue regions.more » « less
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High resolving power ion mobility (IM) allows for accurate characterization of complex mixtures in high-throughput IM mass spectrometry (IM-MS) experiments. We previously demonstrated that pure component IM-MS data can be extracted from IM unresolved post-IM/collision-induced dissociation (CID) MS data using automated ion mobility deconvolution (AIMD) software [Matthew Brantley, Behrooz Zekavat, Brett Harper, Rachel Mason, and Touradj Solouki, J. Am. Soc. Mass Spectrom. , 2014, 25 , 1810–1819]. In our previous reports, we utilized a quadrupole ion filter for m / z -isolation of IM unresolved monoisotopic species prior to post-IM/CID MS. Here, we utilize a broadband IM-MS deconvolution strategy to remove the m / z -isolation requirement for successful deconvolution of IM unresolved peaks. Broadband data collection has throughput and multiplexing advantages; hence, elimination of the ion isolation step reduces experimental run times and thus expands the applicability of AIMD to high-throughput bottom-up proteomics. We demonstrate broadband IM-MS deconvolution of two separate and unrelated pairs of IM unresolved isomers ( viz. , a pair of isomeric hexapeptides and a pair of isomeric trisaccharides) in a simulated complex mixture. Moreover, we show that broadband IM-MS deconvolution improves high-throughput bottom-up characterization of a proteolytic digest of rat brain tissue. To our knowledge, this manuscript is the first to report successful deconvolution of pure component IM and MS data from an IM-assisted data-independent analysis (DIA) or HDMS E dataset.more » « less
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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.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.26434/chemrxiv.12609536.v1
