Protein identification in imaging mass spectrometry through spatially targeted liquid micro‐extractions

Ryan, Daniel; Nei, David; Prentice, Boone M.; Rose, Kristie L.; Caprioli, Richard M.; Spraggins, Jeffrey M.

doi:10.1002/rcm.8042

“…)/360-μm outer diameter (o.d.) fused silica capillary replacing the conventional pipette tip (47), reducing the extraction spot size to ≤1 mm (48,49). However, when using a 50% methanol solvent, as required here for extraction of derivatized sterols, we were unable to achieve this spot size as the surface tension required for a stable liquid microjunction was not attainable.…”

Section: Resultsmentioning

confidence: 96%

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Yutuc

¹

,

Angelini

²

,

Baumert

³

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type andcholesterol 24S-hydroxylaseknockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.

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“…using liquid extraction surface analysis (LESA) technology [54, 55]. The latter has been successfully applied in the study of traumatic brain injuries [56] as well as in mouse brain for the identification of proteins and peptides from MSI experiments [57]. The missing value problem is still a common problem in label free quantitative proteomics.…”

Section: Discussionmentioning

confidence: 99%

Histo-molecular differentiation of renal cancer subtypes by mass spectrometry imaging and rapid proteome profiling of formalin-fixed paraffin-embedded tumor tissue sections

Möginger

¹

,

Marcussen

²

,

Jensen

³

2020

Preprint

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Background: Pathology assessment and differentiation of renal cancer types is challenging due to overlapping histological features of benign and malignant tumors, necessitating highlevel expertise. Mass spectrometry (MS) is an emerging tool for tumor classification of clinical tissue sections by spatial histo-molecular imaging or quantitative microproteomics profiling. Results: We applied MALDI MS imaging (MSI) and LC-MS/MS-based microproteomics technologies to analyze and classify renal oncocytoma (RO, n=11), clear cell renal cell carcinoma (ccRCC, n=12) and chromophobe renal cell carcinoma (CRCC, n=5). Both methods distinguished ccRCC, RO and CCRC with high accuracy in cross-validation experiments (MSI: 93-95%, LC-MS/MS: 100%). Significance: This integrated strategy combining MSI and rapid proteome profiling by LC-MS/MS reveals molecular features of tumor sections and enables cancer subtype classification. Mass spectrometry is a promising complementary approach to current pathological technologies for precise digitized diagnosis of diseases.

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“…/ 360 µm o.d. fused silica capillary replacing the conventional pipette tip (48) reducing the extraction spot size to <1 mm (49,50). However, when using a 50% methanol solvent, as required here for extraction of derivatised sterols, we were unable to achieve this spot size as the surface tension required for a stable liquid micro-junction was not attainable.…”

Section: µLesamentioning

confidence: 96%

Imaging Sterols and Oxysterols in Mouse Brain Reveals Distinct Spatial Cholesterol Metabolism

David

¹

2018

Preprint

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Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatisation in combination with micro-liquid-extraction for surface analysis and liquid chromatography -mass spectrometry to image sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400 µm spot diameter with a limit of quantification of 0.01 ng/mm 2 . It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low abundance and difficult to ionise sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild type and cholesterol 24S-hydroxylase knock-out mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues. SignificanceThe brain is a remarkably complex organ and cholesterol homeostasis underpins brain function. It is known that cholesterol is not evenly distributed across different brain regions, however, the precise map of cholesterol metabolism in the brain remains unclear. If cholesterol metabolism is to be correlated with brain function it is essential to generate such a map. Here we describe an advanced mass spectrometry imaging platform to reveal spatial cholesterol metabolism in situ at 400 µm resolution on 10 µm tissue slices from mouse brain. We mapped, not only cholesterol, but also other biologically active sterols arising from cholesterol turnover in both wild type and mice lacking cholesterol 24-hydroxylase (Cyp46a1), the major cholesterol metabolising enzyme. n g /m m 2

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Protein identification in imaging mass spectrometry through spatially targeted liquid micro‐extractions

Cited by 28 publications

References 45 publications

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Histo-molecular differentiation of renal cancer subtypes by mass spectrometry imaging and rapid proteome profiling of formalin-fixed paraffin-embedded tumor tissue sections

Imaging Sterols and Oxysterols in Mouse Brain Reveals Distinct Spatial Cholesterol Metabolism

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