Laser capture microdissection coupled mass spectrometry (LCM-MS) for spatially resolved analysis of formalin-fixed and stained human lung tissues

Abstract

Background: Haematoxylin 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. Methods: Herein, 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 mm 3) and human lung blood vessels (0.094 mm 3) 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. Results: This 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 mm 3 .

The UIP/IPF fibroblastic focus is a collagen biosynthesis factory embedded in a distinct extracellular matrix

“…For this preparation (a total of 24 samples), we collected tissue volumes of roughly 0.1 mm 3 per sample, which were then processed for MS. This procedure involved detergent-based heat retrieval, physical disruption, and an in-column trypsin digest system to maximize peptide yield, as we did previously ( 18 ).…”

“…Recently, our group has developed a laser capture microdissection-coupled mass spectrometry (LCM-MS) technique to characterize the ECM of fixed and stained human lung tissue ( 18 ), which we now apply to the FF and surrounding tissue. Here, we present a comprehensive molecular tissue atlas that defines cell surface and ECM proteins and conduct pathway analysis of histologically defined regions of the UIP/IPF lung in an effort to understand its pathogenesis.…”

IL-13 deficiency exacerbates lung damage and impairs epithelial-derived type 2 molecules during nematode infection

“…Samples from slides containing whole lung tissue sections were scraped excluding major blood vessels and processed as previously described ( Herrera et al, 2020 ). Peptides were evaluated by liquid chromatography coupled tandem mass spectrometry using an UltiMate 3000 Rapid Separation LC system (Dionex Corporation) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific).…”

The liver metastatic niche: modelling the extracellular matrix in metastasis

“…There are technical limitations in isolating smaller early metastases, which are otherwise outweighed by the bulk of normal liver tissue in dECM preparations. Techniques such as laser capture microdissection in combination with mass spectrometry have been used to isolate and profile micrometastases and ECM, and could be useful ( Herrera et al, 2020 ; Elbouchtaoui et al, 2018 ). This technique can also be used to profile different parts of the tumour, such as the invasive front, to include spatial information ( Carnielli et al, 2018 ).…”