Establishment of a targeted proteomics method for the quantification of collagen chain: Revealing the chain stoichiometry of heterotypic collagen fibrils in sea cucumber

Collagen is the most abundant and important structural biomacromolecule in sea cucumbers. The sea cucumber collagen fibrils were previously confirmed to be heterotypic, nevertheless, the stoichiometry of collagen α-chains governing the complexity of collagen fibrils is still poorly understood. Herein, four representative collagen α-chains in sea cucumber including two clade A fibrillar collagens, one clade B fibrillar collagen, and one fibril-associated collagen with interrupted triple helices were selected. After the screening of signature peptides and optimization of multiple reaction monitoring (MRM) acquisition parameters including fragmentation, collision energy, and ion transition, a feasible MRM-based method was established. Consequently, the stoichiometry of the four collagen chains was determined to be approximately 100:54:3:4 based on the method. The assembly forms of sea cucumber collagen fibrils were further hypothesized according to the chain stoichiometry. This study facilitated the quantification of collagen and understanding of the collagen constituents in sea cucumber.