Journal: Nature Communications

Article Title: Robust collection and processing for label-free single voxel proteomics

doi: 10.1038/s41467-024-54643-x

Figure Lengend Snippet: a There are three steps for wcSOP-MS: wet cap collection, SOP processing, and LC-MS/MS analysis. A tissue slide mounted on PEN membrane is cut by laser and a single tissue voxel is catapulted to a dome-shaped PCR tube cap prefilled with a cocktail of buffer containing DDM surfactant for tissue lysis and a mixture of Lys-C and trypsin protease for protein digestion. Single voxels are incubated at 75 °C for 1 h (tissue lysis and protein denaturation) and 37 °C for overnight (protein digestion) on the cap at a hanging droplet position to have full interaction between the voxel and the cocktail buffer. After voxel processing, the digested peptides are transferred to the bottom of the tube by centrifugation at 2000 × g for 5 min. Prior to LC-MS analysis, the cap of the PCR tube is removed, and the tube is inserted into a sample vial to avoid sample transfer loss. Single voxels are analyzed by using a standard LC-MS platform for quantitative proteomic analysis. The freely-available open-source MaxQuant and DIA-NN software tools are used for DDA and DIA label-free quantitation, respectively. b Comparison of the number of identified protein groups (No. of protein groups) between DDA and DIA for single spleen tissue voxels at a size of 200 µm × 200 µm × 10 µm ( n = 3 biological replicates; data are presented as mean values ± SD). c Venn diagram of protein overlap between DDA and DIA for single spleen tissue voxels at a size of 200 µm × 200 µm × 10 µm. d Proteome dynamic range for DDA and DIA quantification of single spleen tissue voxels at a size of 200 µm × 200 µm × 10 µm. e Comparison of the coefficient of variations (CVs) in protein abundance between DDA and DIA for analysis of single spleen tissue voxels at a size of 200 µm × 200 µm × 10 µm with 4 biological replicates. Source data are provided as a file.

Article Snippet: Prior to LCM experiments, for wet collection a dome-shaped cap of PCR tube (0.2 ml of PCR individual tubes with domed cap, GeneMate) was prepopulated with a 25 μL buffer droplet, and for dry collection a PCR tube cap with adhesive polymer membrane was used.

Techniques: Liquid Chromatography with Mass Spectroscopy, Membrane, Lysis, Incubation, Centrifugation, Software, Quantitation Assay, Comparison, Quantitative Proteomics