Background
Despite the utility of mass spectrometry imaging (MSI) for spatial proteomics, peptide delocalization during sample preparation compromises the resolution of peptide images and their association with histological features. Deep visual proteomics is a recently developed strategy that overcomes these challenges, and we investigated several aspects of this technique (laser microdissection with LC-MS/MS) to complement our MSI experiments. Single cells from specific regions of mouse tissues were obtained from laser microdissection and a bottom-up proteomics approach was performed.
Methods
Control and fibrotic mouse lungs were inflated in situ with 4 % paraformaldehyde neutral buffer and processed as formalin-fixed paraffin embedded (FFPE) tissue. Tissues were sectioned (6 µm) and water mounted on polyethylene naphthalate (PEN) membrane slides. Sections were dewaxed, rehydrated and antigen retrieval performed with citric acid, followed by a hematoxylin counter stain for visualisation. Alveolar regions were obtained using a Leica LMD 7 laser microdissection microscope, with 20, 50, 400, and 1000 cells from control tissue and 400 cells from diseased samples (non fibrotic or fibrotic regions) subsequently denatured and trypsin digested. Samples were vacuum dried and reconstituted before data-independent acquisition parallel accumulation-serial fragmentation (dia-PASEF) was performed on a Bruker timsTOF fleX. Data files were processed with Spectronaut using default settings.
Results
Approximately 3200 proteins were identified from the 1000 cells control and 1750 proteins identified from the 20 cells. Among the 400 cell proteins were found in each group. There were unique proteins specifically only detectable in each of the sample type.
Conclusions
Laser microdissection LC-MS/MS spatial proteomics was enabled precise region-specific proteomic experiments using low sample quantity down to 20 cells from FFPE tissues. Experimental output suggesting of samples containing 400 cells, was sufficient to differentiate the protein abundance in different sample among the control sample, ‘non fibrotic’ region, and ‘fibrotic’ region of the diseased lungs.