A recommended and verified procedure for <i>in situ</i> tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Judd, Audra M.; Gutierrez, Danielle B.; Moore, Jessica L.; Patterson, Nathan Heath; Yang, Junzhong; Romer, Carrie; Norris, Jeremy L.; Caprioli, Richard M.

doi:10.1002/jms.4384

Cited by 37 publications

(41 citation statements)

References 38 publications

(50 reference statements)

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“…On-tissue digestion was performed as described previously (Judd et al, 2019). Briefly, the sections were sprayed with trypsin (15 ng/µL in 10% ACN in 100 mM ammonium bicarbonate, pH 8) using a TM Sprayer (HTX Technologies, Carrboro, NC, USA) modified with a syringe pump at 8 μL/min (Harvard Apparatus, Holliston, MA, USA).…”

Section: On-tissue Digestion and Maldi Matrix Applicationmentioning

confidence: 99%

“…In addition, soluble proteins are more favored in the MALDI process (Seeley and Caprioli, 2008); however, many important proteins are neither small nor soluble. To overcome these limitations, in situ digestion protocols were developed that typically yield peptides between 400 and 3500 Da; a range in which instrumental sensitivity is higher than that for intact proteins (Groseclose et al, 2007;Judd et al, 2019;Lemaire et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry

Wang

Ryan²,

Schey³

2020

Preprint

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Imaging mass spectrometry (IMS) enables targeted and untargeted visualization of the spatial localization of molecules in tissues with great specificity. The lens is a unique tissue that contains fiber cells corresponding to various stages of differentiation that are packed in a highly spatial order. The application of IMS to lens tissue localizes molecular features that are spatially related to the fiber cell organization. Such spatially resolved molecular information assists our understanding of lens structure and physiology; however, protein IMS studies are typically limited to abundant, soluble, low molecular weight proteins. In this study, a method was developed for imaging low solubility cytoskeletal proteins in the lens; a tissue that is filled with high concentrations of soluble crystallins. Optimized tissue washes combined with on-tissue enzymatic digestion allowed successful imaging of peptides corresponding to known lens cytoskeletal proteins. The resulting peptide signals facilitated segmentation of the bovine lens into molecularly distinct regions. A sharp intermediate filament transition from vimentin to lens-specific beaded filament proteins was detected in the lens cortex. MALDI IMS also revealed the region where posttranslational myristoylation of filensin occurs and the results indicate that truncation and myristoylation of filensin starts soon after filensin expression increased in the inner cortex. From intermediate filament switch to filensin truncation and myristoylation, multiple remarkable changes occur in the narrow region of lens cortex. MALDI images delineated the boundaries of distinct lens regions that will guide further proteomic and interactomic studies.

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Section: On-tissue Digestion and Maldi Matrix Applicationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry

Wang

Ryan²,

Schey³

2020

Preprint

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“…11,12 IMS offers an untargeted mapping capability for hundreds to thousands of molecules in a single experiment. Moreover, IMS provides the analytical flexibility to investigate different classes of biomolecules including small metabolites, 13,14 lipids, 15,16 drugs, 17,18 glycans, 19,20 peptides, 21,22 and proteins. 23,24 However, IMS faces challenges of dynamic range, peak capacity, and the ability to structurally identify as defined by the Mason-Schamp equation.…”

Section: Introductionmentioning

confidence: 99%

Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspective

et al. 2020

Self Cite

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Imaging mass spectrometry (IMS) technologies are capable of mapping a wide array of biomolecules in diverse cellular and tissue environments. IMS has emerged as an essential tool for providing spatially targeted molecular information due to its high sensitivity, wide molecular coverage, and chemical specificity. One of the major challenges for mapping the complex cellular milieu is the presence of many isomers and isobars in these samples. This challenge is traditionally addressed using orthogonal liquid chromatography (LC)-based analysis, though, common approaches such as chromatography and electrophoresis are not able to be performed at timescales that are compatible with most imaging applications. Ion mobility offers rapid, gas-phase separations that are readily integrated with IMS workflows in order to provide additional data dimensionality that can improve signal-to-noise, dynamic range, and specificity. Here, we highlight recent examples of ion mobility coupled to IMS and highlight their importance to the field.

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“…After the pioneering phase, challenging technical aspects of this approach, such as the interference of hemoglobin and the stability of the samples, were overcome [4,5]. Furthermore, recent technical improvements related to the increased lateral resolution that can be achieved by MALDI-TOF-MS instrumentation enable the detection of small cell subpopulations based on their different protein profiles (i.e., profiles of single cell types), even within regions that are indistinguishable at the microscopic level, highlighting how molecular imaging can be combined with traditional pathology to generate protein signatures and build classification models [6,7,8,9]. Moreover, we have reported that MALDI-MSI is able to distinguish benign and malignant cases in different cytological thyroid specimens [1,2,3].…”

Section: Introductionmentioning

confidence: 99%

MALDI-MSI as a Complementary Diagnostic Tool in Cytopathology: A Pilot Study for the Characterization of Thyroid Nodules

Capitoli

Piga

Galimberti

et al. 2019

Cancers

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The present study applies for the first time as Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging (MSI) on real thyroid Fine Needle Aspirations (FNAs) to test its possible complementary role in routine cytology in the diagnosis of thyroid nodules. The primary aim is to evaluate the potential employment of MALDI-MSI in cytopathology, using challenging samples such as needle washes. Firstly, we designed a statistical model based on the analysis of Regions of Interest (ROIs), according to the morphological triage performed by the pathologist. Successively, the capability of the model to predict the classification of the FNAs was validated in a different group of patients on ROI and pixel-by-pixel approach. Results are very promising and highlight the possibility to introduce MALDI-MSI as a complementary tool for the diagnostic characterization of thyroid nodules.

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A recommended and verified procedure for in situ tryptic digestion of formalin‐fixed paraffin‐embedded tissues for analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry

Cited by 37 publications

References 38 publications

Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry

Localization of the Lens Intermediate Filament Switch by Imaging Mass Spectrometry

Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspective

MALDI-MSI as a Complementary Diagnostic Tool in Cytopathology: A Pilot Study for the Characterization of Thyroid Nodules

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