MALDI MSI\nProtocol for Spatial Bottom-Up Proteomics\nat Single-Cell Resolution

Matrix-assisted laser desorption/ionization (MALDI) mass\nspectrometry\nimaging (MSI) started with spatial mapping of peptides and proteins.\nSince then, numerous bottom-up protocols have been developed. However,\nachievable spatial resolution and sample preparation with many wet\nsteps hindered the development of single cell-level workflows for\nbottom-up spatial proteomics. This study presents a protocol optimized\nfor MALDI-MSI measurements of single cells within the context of their\n2D culture. Sublimation of CHCA, followed by a dip in ice-cold ammonium\nphosphate monobasic (AmP), produced peptide-rich mass spectra while\nmaintaining matrix crystal sizes around 400 nm. This enables MALDI-MSI\nimaging of proteins in single cells grown on an ITO slide with a throughput\nof approximately 7800 cells per day. 89 peptide-like features corresponding\nto a single MDA-MB-231 breast cancer cell were detected. Furthermore,\nby combining the MALDI-MSI data with LC-MS/MS data obtained on cell\npellets, we have successfully identified 24 peptides corresponding\nto 17 proteins, including actin, vimentin, and transgelin-2.