Dataset Information

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans.

ABSTRACT: The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

SUBMITTER: Pandey S

PROVIDER: S-EPMC8232303 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans.

Pandey Shashank S Tuma Zdenek Z Smrhova Tereza T Cedikova Miroslava M Macanova Tereza T Chottova Dvorakova Magdalena M

Pharmaceutics 20210615 6

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm<sup>2</sup> with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA ...[more]

PMID: 34203686

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Project description:BackgroundHaematoxylin and eosin (H&E)-which respectively stain nuclei blue and other cellular and stromal material pink-are routinely used for clinical diagnosis based on the identification of morphological features. A richer characterization can be achieved by laser capture microdissection coupled to mass spectrometry (LCM-MS), giving an unbiased assay of the proteins that make up the tissue. However, the process of fixing and H&E staining of tissues provides challenges with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique to analyse H&E-stained, formalin-fixed paraffin-embedded (FFPE) tissues.MethodsHerein, we utilize heat extraction, physical disruption, and in column digestion for the analysis of H&E stained FFPE tissues. Micro-dissected morphologically normal human lung alveoli (0.082 mm3) and human lung blood vessels (0.094 mm3) from FFPE-fixed H&E-stained sections from Idiopathic Pulmonary Fibrosis (IPF) specimens (n = 3 IPF specimens) were then subject to a qualitative and then quantitative proteomics approach using BayesENproteomics. In addition, we tested the sensitivity of this method by processing and analysing a range of micro-dissected human lung blood vessel tissue volumes.ResultsThis approach yields 1252 uniquely expressed proteins (at a protein identification threshold of 3 unique peptides) with 892 differentially expressed proteins between these regions. In accord with prior knowledge, our methodology approach confirms that human lung blood vessels are enriched with smoothelin, CNN1, ITGA7, MYH11, TAGLN, and PTGIS; whereas morphologically normal human lung alveoli are enriched with cytokeratin-7, -8, -18, -19, 14, and -17. In addition, we identify a total of 137 extracellular matrix (ECM) proteins and immunohistologically validate that laminin subunit beta-1 localizes to morphologically normal human lung alveoli and tenascin localizes to human lung blood vessels. Lastly, we show that this micro-proteomics technique can be applied to tissue volumes as low as 0.0125 mm3.ConclusionHerein we show that our multistep sample preparation methodology of LCM-MS can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues.

Project description:The etiology of Type 1 Diabetes (T1D) remains elusive. Enzymatically isolated and cultured (EIC) islets cannot fully reflect the natural protein composition and disease process of in vivo islets, because of the stress from isolation procedures. In order to study islet protein composition in conditions close to the natural environment, we performed proteomic analysis of EIC islets, and laser capture microdissected (LCM) human islets and acinar tissue from fresh-frozen pancreas sections of three cadaveric donors. 1104 and 706 proteins were identified from 6 islets equivalents (IEQ) of LCM islets and acinar tissue, respectively. The proteomic profiles of LCM islets were reproducible within and among cadaveric donors. The endocrine hormones were only detected in LCM islets, whereas catalytic enzymes were significantly enriched in acinar tissue. Furthermore, high overlap (984 proteins) and similar function distribution were found between LCM and EIC islets proteomes, except that EIC islets had more acinar contaminants and stress-related signal transducer activity proteins. The comparison among LCM islets, LCM acinar tissue and EIC islets proteomes indicates that LCM combined with proteomic methods enables accurate and unbiased profiling of islet proteome from frozen pancreata. This paves the way for proteomic studies on human islets during the progression of T1D.SignificanceThe etiological agent triggering autoimmunity against beta cells in Type 1 diabetes (T1D) remains obscure. The in vitro models available (enzymatically isolated and cultured islets, EIC islets) do not accurately reflect what happens in vivo due to lack of the natural environment where islets exist and the preparation-induced changes in cell physiology. The importance of this study is that we investigated the feasibility of laser capture microdissection (LCM) for the isolation of intact islets from frozen cadaveric pancreatic tissue sections. We compared the protein profile of LCM islets (9 replicates from 3 cadaveric donors) with that of both LCM acinar tissues (6 replicates from the same 3 cadaveric donor as LCM islets) and EIC islets (at least 4 replicates for each sample with the same islets equivalents) by using proteomics techniques with advanced instrumentation, nanoLC-Q Exactive HF Orbitrap mass spectrometry (nano LC-MS/MS). The results demonstrate that the LCM method is reliable in isolating islets with an intact environment. LCM-based islet proteomics is a feasible approach to obtain good proteome coverage for assessing the pathology of T1D using cadaveric pancreatic samples, even from very small sample amounts. Future applications of this LCM-based proteomic method may help us understand the pathogenesis of T1D and identify potential biomarkers for T1D diagnosis at an early stage.

Dataset Information

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans.

Publications

Laser Capture Microdissection Coupled Capillary Immunoassay to Study the Expression of PCK-2 on Spatially-Resolved Islets of Rat Langerhans.

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