Robust collection and processing for label-free single voxel proteomics.

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures tissue heterogeneity, precluding proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single microscale tissue voxels and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single voxels dissected by LCM to the tube cap and SOP voxel processing in the same collection cap. This method enables reproducible, label-free quantification of approximately 900 and 4600 proteins for single voxels at 20 µm × 20 µm × 10 µm (~1 cell region) and 200 µm × 200 µm × 10 µm (~100 cell region) from fresh frozen human spleen tissue, respectively. It can reveal spatially resolved protein signatures and region-specific signaling pathways. Furthermore, wcSOP-MS is demonstrated to be broadly applicable for OCT-embedded and FFPE human archived tissues as well as for small-scale 2D proteome mapping of tissues at high spatial resolutions. wcSOP-MS may pave the way for routine robust single voxel proteomics and spatial proteomics.