Tmt 6 plex labeling kit

The ExoQuick ULTRA exosome isolation kit was purchased from SBI System Bioscience (Palo Alto, CA, USA). Anti-L1CAM biotinylated antibody was purchased from R&D Systems (Minneapolis, MN, USA). The human CD81 (Cluster of Differentiation 81) antigen ELISA kit was purchased from CUSABIO (Wuhan, China). ELISA kits for C7 (Complement component 7), FERMT3 (Fermitin Family Member 3), CAP1 (Adenylyl cyclase-associated protein 1), ENO1 (Enolase 1), and ZYX (Zyxin) were purchased from CLOUD-CLONE (Wuhan, China). Albumin/IgG removal kits were purchased from Merk (Shanghai, China). TMT 6-plex labeling kit, MicroBCA protein quantification kit, Streptavidin Plus UltraLink™ Resin, M-PER Mammalian Protein Extraction Reagent, 1M TEAB, and 50% Hydroxylamine were purchased from Thermo Scientific (Rockford, IL, USA). Sequence grade trypsin was purchased from Promega (Madison, WI, USA). Dithiothreitol and indole-3-acetic acid were purchased from GE Healthcare (Shanghai, China).

R. wratislaviensis C31-06 cells were prepared in Aib-inducible or non-inducible conditions. The enrichment medium with 0.05% (w/v) glucose was used for induction, and medium without Aib was used for non-induction. The addition of 0.05% glucose helped the growth of C31-06 cells without inhibiting the Aib-degrading activity. R. wratislaviensis C31-06 was cultivated in each medium for 38.5 h. Sample preparation and proteome analysis were performed by previously reported with slight modification^{21 (link)}. Cells were disrupted with 0.10 mm beads using a multibeads shocker (Yasui Kikai, Osaka, Japan), and digested with trypsin. The six tryptic digests, including triplicate Aib-induction and non-induction samples, were labeled using a tandem mass tag (TMT) 6-plex labeling kit (Thermo Fisher Scientific, MA, USA), and then dissolved in 60 μL 0.1% (v/v) formic acid. LC-MS/MS was conducted on an Ultimate 3000 HPLC and an LTQ Orbitrap Velos Mass Spectrometer (Thermo Fisher Scientific) with a monolithic silica capillary column (500 cm long, 0.1 mm ID)^{44 (link)}.

The desalted samples were dissolved in 50 μL of TEAB. The dissolved peptide solution was labeled using a TMT 6-plex labeling kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol with a slight modification. The TMT-labeling reagents were dissolved in 41 μL of acetonitrile and mixed with 10 μL of TMT-labeling reagents to 100 μL. Each digested peptide solution was mixed with 10 μL of TMT-labeling reagents as described in Fig. 1. The tryptic digests of the lag, early log, middle log, late log, and stationary phases were labeled TMT-126, TMT-127, TMT-128, TMT-129, and TMT-130, respectively. In addition, an equal volume of all samples was mixed, and the mixture was labeled TMT-131 as an internal standard. After the labeling reaction, performed at room temperature for 60 min, the reactants were quenched by adding 8 μL of 5% hydroxylamine and lyophilized after mixing with 30 μL of each reactant. The dried samples were resolved in 30 μL of 0.1% formic acid.