High‐Throughput Single‐Cell‐Resolved Spatial Proteomics Enabled by an Ordered Colloidal Crystal Column

, 10-fold higher than that of commonly used sub-2-µm particle packed columns, enabling robust and rapid peptide separation. With such a column, high-throughput nanoLC-MS analysis was achieved, as demonstrated by the identification of 5942 and 4388 proteins from 250 pg HeLa digests using 5- and 2-min gradients, respectively. More notably, the column exhibited exceptional performance in single-cell spatial proteomics, enabling the identification of up to 2304 proteins from a single hepatocyte slice within only a 5-min gradient. Even under an ultrarapid 2-min gradient, up to 1292 proteins were identified from single-cell slices, which is 16 times faster than conventional methods. All these results demonstrated great promise of the colloidal crystal column for high-throughput spatial proteomics with single-cell resolution.