Spatial Distribution of Silk Proteins in the Silk Gland of Bombyx mori

Natural silk is a highly successful structural material owing to the precise composition and ordered organization of its constituent proteins. However, the microscale spatial organization of silk proteins within the silk gland lumen remains poorly understood. Here, we established a laser microdissection coupled with the liquid chromatography-tandem mass spectrometry (LM-LC-MS/MS) strategy to characterize layer-resolved protein composition across the silk gland lumen of DaZao. A total of 248 proteins were identified from 1334 peptides, revealing distinct yet continuous compositional gradients along the radial axis. Fibroin heavy chain, fibroin light chain, and fibrohexamerin were enriched in inner regions, whereas sericin and low-abundance silk-associated proteins increased toward the periphery. Notably, these proteins exhibited overlapping rather than sharply separated distributions across conventional fibroin and sericin regions. These findings reveal a dynamic permeable layering pattern during silk formation and provide new insights into silk fiber assembly.