Easy n lc chromatograph

A His‐tag affinity pull‐down assay was performed to identify the protein partners of TLP‐1 in the shell matrices of M. coruscus using the protocol of Jiang et al. [33]. Ni‐NTA beads (Sangon, Shanghai, China) were used for binding rTLP‐1 at the His₆ tag contained in the sequence, and the beads were washed with binding buffer (20 mm Tris/HCl, 150 mm NaCl and 10 mm imidazole at pH 8.0) and then incubated for 4 h with total proteins extracted from the shell matrices of M. coruscus at 4 °C and then washed with elution buffer (20 mm Tris/HCl, 300 mm NaCl and 300 mm imidazole at pH 8.0). The eluted fractions were analysed by LC‐MS/MS after digestion by trypsin. The LC‐MS/MS experiments were performed on a Q‐Exactive Plus MS spectrometer coupled with an Easy‐nLC chromatograph (Thermo Scientific, Waltham, Massachusetts, USA). The MS data were analysed using maxquant software (version 1.6.1.0., Max‐Planck Institute of Biochemistry, Netherlands, Germany) and searched against the mantle transcriptome database of M. coruscus (Accession: SRX792025) [6]. The database search results were filtered and exported based on a < 1% false discovery rate (FDR) at the peptide‐spectrum‐matched level and protein level.