Smart digest trypsin kit

Tryptic digestion was performed using Thermo Scientific™ SMART Digest™ trypsin kit and the Thermo Scientific™ KingFisher Duo Prime system following the manufacturer's protocol and as previously described [15 (link)]. Briefly, 100 μg of the sample (2 mg/mL) was diluted with 150 μL of SMART digest buffer in a Thermo Scientific™ KingFisher Deepwell 96-well plate. Following a wash step, magnetic beads obtained from 15 μL of magnetic SMART bead solution were added to the sample and incubated at 70 °C for 60 min. Following digestion, disulfide bonds were reduced by incubating the samples in 10 mM DTT for 20 min at room temperature, followed by alkylation with 3 mM IAA at room temperature for 15 min in the dark. Subsequently, the samples were evaporated to dryness and resuspended in 0.1% FA in H₂O to a final concentration of 1 μg/μL.

Live T-47D cells were plated on 6 cm petri dishes supplemented with RPMI media and incubated for 24 h. Cells were then treated with probe 1i (100 µM to 2 mM), CuBr (100 µM to 2 mM), and 1% acetonitrile for 2 h. After 2 h, cells were washed 3 times with cold PBS and lysed using RIPA buffer (50 mM Tris HCl [pH 8], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors. Lysates were centrifuged 6500x g, 10 min at 4 °C, and soluble lysate was collected. To 100 µg of lysate in 100 µL of PBS buffer were treated with 50 µL of 100 mM TBTA in water, 50 µL of freshly prepared 100 mM ascorbic acid in water, 50 µL of 50 mM of CuSO₄ in water, and 2 µL of 10 mM Cy5 azide in DMSO. The reaction was stirred for 1 h and acetone precipitated, followed by analysis of proteins through in-gel fluorescence imaging and Coomassie blue staining. Samples were loaded on a Novex WedgeWell 4–20% Tris-Glycine gel. Gel was run in Tris-glycine running buffer at 180 V. The gel was then stained with Coomassie brilliant blue for 1 h and destained overnight. For proteomics analysis, 100 µg of live-cell derived lysates were digested using SMART Digest™ Trypsin Kit by Thermo Scientific. Proteomics analysis was done according to the general protocol described above.

The IP procedure was performed as described previously 27 (link). Accordingly, cells expressing pCDNA3-2×Flag-FOXP3 or pCDNA3-2×Flag were lysed in 1×RIPA lysis buffer supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, #78430), followed by centrifuging at 13,000 rpm for 15 min. The supernatant was incubated with anti-Flag agarose beads (Sigma-Aldrich, #A2220) at 4°C for 2 h. After washing 5 times with 1×RIPA lysis buffer, the Flag-FOXP3-associated protein complex was separated in a 10% SDS-PAGE gel and then stained with Coomassie Brilliant Blue R 250 (Thermo Fisher Scientific, #20278) for 15 min. Protein bands were digested using a SMART Digest Trypsin Kit (Thermo Fisher Scientific, #60109101), followed by mass spectrometry analysis as described previously 27 (link).

SMART Digest Trypsin Kit (Thermo Fisher Scientific) was used for digesting 50 µL of cross-linked sample. 150 µL of SMART Digest buffer containing 5 µg of beads were added and the resulting solutions were incubated at 70 °C for 3 h. After cooling down the samples were centrifuged and the supernatant was collected. Peptides were further incubated with DTT (4 mM) for 30 min at 56 °C and iodoacetamide (8 mM) for 20 min at room temperature in the dark. Alkylation was quenched by adding 4 mM of DTT. Finally, TFA was added to the samples.

After 4 weeks, all previous samples stored in -80 °C were digested using the SMART Digest^™ Trypsin Kit (Thermo Scientific^™, San Jose, CA). Briefly, 100 μg of monoclonal antibody was digested with trypsin beads. The filtered solution was mixed with dithiothreitol (Fisher, Hampton, NH) for 30 min to break inter and intra disulfide bonds in the heavy chains (HC) and light chains (LC). Iodoacetamide (Sigma, St. Louis, MO) was added and left in the dark for 30 min to block any free cysteine residues. The reaction was stopped by the addition of trifluoroacetic acid.

Exosomes were lysed by radioimmunoprecipitation assay (RIPA) buffer to extract the exosomal proteins. Then, the proteins were digested by trypsin (SMART Digest Trypsin Kit, Thermo Fisher Scientific; Waltham, MA), desalted (Millipore® Ziptips Micro-C18; Sigma-Aldrich, Milwaukee, WI), purified (SOLAµ™ SPE Plates; Thermo Fisher Scientific; Waltham, MA), and dissolved in 0.1% formic acid for LC-MS/MS analysis (LTQ Orbitrap Velos, Thermo Scientifics; Waltham, MA) (service provided by the Mass Core Facility of Genomics Research Center, Academia Sinica). The acquired proteomic raw data files were then applied to search against a UniProt human protein database by using PEAKS Studio 7.5 (Bioinformatics Solutions, Waterloo, Ontario, Canada). The settings in PEAKS Studio 7.5 combined with UniProt for searching the protein database were as follows: enzyme set as trypsin with a maximum of two missed cleavage site precursor and fragment mass tolerance of 20 ppm and 0.8 Da, respectively. Finally, spectral counts obtained from each peptide were normalized to the total spectral counts recorded for all peptides in a sample.