ABSTRACT Haematoxylin and eosin (H&amp;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&amp;E staining of tissues is poorly compatible with standard sample preparation methods for mass spectrometry, resulting in low protein yield. Here we describe a microproteomics technique optimized to analyze H&amp;E-stained, formalin-fixed paraffin-embedded (FFPE) tissues. We advance our methodology by combining 3 techniques shown to individually enhance protein yields (heat extraction, physical disruption, and in column digestion) into one optimized pipeline for the analysis of H&amp;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 section from Idiopathic Pulmonary Fibrosis (IPF) specimens were then subject to comparative proteomics using this methodology. This approach yielded 1252 differentially expressed proteins including 137 extracellular matrix (ECM) proteins. In addition, we offer proof of principal that MS can identify distinct, characteristic proteomic compositions of anatomical features within complex tissues.

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