Autoflex 2

Peptides were incubated at 37°C in 250 μl of human serum (Sigma Aldrich) for different periods of time. Samples were collected and peptide degradation stopped by freezing. Peptides were extracted from samples using the Proteo Miner Protein Enrichment System (Bio-Rad). Percentage of intact peptide was estimated by mass spectrometry (MS) using MALDI-TOFF as described previously[19 (link)] (Bruker Autoflex II) following their protocol. Measurements were performed in triplicate. MS data were analysed using the software Cliprot tools, Felx analysis, Bruker.

HT-29 cells were lysed with NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 1 mM DTT, and 0.5 mM PMSF) on ice for 30 min and centrifuged at 20,400 × g for 10 min at 4 °C. The supernatants were used as the whole-cell extracts of HT-29 cells. The extracts were incubated with empty or fucoxanthinol-fixed beads at 4 °C for 4 h. The beads were then washed three times with binding buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1% NP-40). The proteins binding to the beads were eluted with Laemmli SDS sample buffer, subjected to 12% SDS-PAGE, and detected by silver staining. Each gel slice containing a fucoxanthinol-binding protein was subjected to in-gel digestion with Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA). The resulting peptides were analyzed by Autoflex II (Bruker Daltonics, Billerica, MA, USA), and proteins were identified by peptide mass fingerprinting.
The recombinant His-tagged uS7 protein (His-uS7, ab137146, Abcam) was incubated with empty or fucoxanthinol-fixed beads at 4 °C for 4 h. The beads were then washed three times with binding buffer. His-uS7 binding to the beads was eluted with Laemmli SDS sample buffer, subjected to 12% SDS-PAGE, and detected by western blotting.