Abstract

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.

Highlights

  • 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

  • Conclusion: we show that our multistep sample preparation methodology of laser capture microdissection coupled to mass spectrometry (LCM-Mass spectrometry (MS)) can identify distinct, characteristic proteomic compositions of anatomical features within complex fixed and stained tissues

  • Laser capture microdissection coupled to mass spectrometry (LCM-MS) is a method currently being optimized for microproteomics to determine regional tissue differences [3]

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Summary

Introduction

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. Mass spectrometry (MS) proteomics is a powerful tool to systemically identify and quantify proteins in complex biological samples The utility of this method is maximized when performed with spatial resolution to report on the composition and function of specific regions of tissue. Common protocols for bottom-up proteomics (i.e. based on detection of peptide protein fragments) require sample homogenization and digestion, resulting in a loss of any information regarding protein localization and spatial relationships. To this end, laser capture microdissection coupled to mass spectrometry (LCM-MS) is a method currently being optimized for microproteomics to determine regional tissue differences [3]. We describe and examine the performance of a protocol for LCM-MS analysis of FFPE sections of human lung tissue that were haematoxylin and eosin (H&E) stained

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