Click-iT Protein Enrichment Protocol

The CuAAC reaction was set up using the Click-iT Protein Enrichment
Kit (Invitrogen). In short, 100 μL of alkyne bead slurry was
washed with 1 mL ultrapure H₂O, after which 250 μL
of concentrated medium, 250 μL of urea buffer, 500 μL
of 2× catalyst solution, and 1× CPIs were added. This was
incubated for 16–20 h at room temperature while rotating, after
which the beads were washed with 1 mL ultrapure H₂O. Next,
reduction and alkylation of the bound proteins were done by incubating
the beads with 10 mM DTT in 500 μL of SDS buffer for 15 min
while shaking, followed by incubation with 50 mM iodoacetamide (IAA)
in 500 μL of SDS buffer for 30 min while shaking in the dark.
The beads were transferred to spin columns and washed with 20 mL of
SDS buffer, 20 mL of 8 M urea in 100 mM Tris, pH 8, 20 mL 20% isopropanol,
20 mL 20% acetonitrile, and 5 mL of PBS. The bound proteins were digested
by resuspending the beads in 200 μL of freshly prepared digestion
buffer (2 M Urea, 100 mM Tris-HCl pH 8, 100 mM DTT) with 0.5 μg
of mass spec-grade trypsin (Promega) and overnight incubation at room
temperature while shaking. The digest was desalted and concentrated
on C18 StageTips without acidification.^{24 (link)} Peptide labeling was done by dimethyl labeling,^{27 (link)} and StageTips were stored at 4 °C until measurement
by LC-MS/MS.