Polyacrylamide desalting column

To obtain small molecules in the CM, the pooled CM was applied to the 15ml Pierce protein concentrator (ThermoFisher Scientific, with the cutoff molecular weight of 3KD, and centrifuged at 2,000xg for 30min. The bottom fraction containing molecules below 3KD was lyophilized and dissolved in ddH₂O (1/8 the original volume). The concentrated CM<3KD was desalted and partially purified with 5ml polyacrylamide desalting column (ThermoFisher Scientific) by gravity following the manufacturer’s protocol. Fractions obtained from desalting (10% vol/vol) was then tested in T cell proliferation assay. The positive fractions were pooled and separated by HPLC with ultrapure water as the mobile phase isocratically on a reverse phase C18 analytical (4.6×250 mm, 5μm) column (Guangzhou Research&Creativity Biotechnology Co., Ltd) at the flow rate of 0.3 mL min^-1. The UV detector was set at 210 nm.

Site-specific labeling of αS was performed in an αS variant with a single engineered cysteine at position 122 (αS N122C). This variant was expressed and purified as described above but including 5 mM DTT in all purification steps. The protein was labeled with maleimide-modified Alexa Fluor 488 (AF488) (Invitrogen, Carlsbad, USA) for 15–20 h at 4 °C in the dark. After quenching the reaction with 10 mM DTT, free unreacted dye in the protein solution was subsequently separated using a P10 desalting column (GE Healthcare, Waukesha, USA), and the labeled protein solution was flash frozen with liquid nitrogen and stored at −80 °C. The different peptides, PSMα3, dPSMα3, and LL-37, were labeled at a single engineered cysteine at the N-terminus with maleimide-modified Atto647N (ATTO-TEC, Siegen, Germany). The same labeling and purification strategy were followed as for αS, although in this case the unreacted free dye was removed from the protein solution using a polyacrylamide desalting column (Thermo Fisher Scientific, Waltham, USA). Two cleaning steps were required to remove completely the free dye from the labeled peptide solution.