Proximity labeling and enrichment of biotinylated protein

Reagents

SILAC DMEM medium (Thermofisher, 88364)

L-Arginine-HCl, 13C6, 15N4 for SILAC (Thermofisher, 89990)

L-Lysine-2HCl, 13C6, 15N2 for SILAC (Thermofisher, 88209)

Biotinyl Tyramide (Iris-Biotech, 41994-02-9)

Hydrogen peroxide (Sigma, H1009)

Sodium azide (Sigma, S2002)

Sodium ascorbate (Sigma, 11140)

Trolox (Sigma, 238813)

Benzonase (Millipore, 1016970010)

2-D quant kit (GE healthcare, 80648356)

TCEP (Sigma, C4706)

Iodoacetamide (Sigma, A3221)

Streptavidin magnetic beads (Thermofisher, 88816)

TEAB (Thermofisher, 90114)

Formic acid (Sigma, 33015)

1. Before the labeling experiment, U2OS cells were passaged in (Lys0, Arg0) or heavy (Lys8, Arg10) DMEM medium for 5 generations. For each condition, a confluent 10-cm dish of cells (1-2 x 10⁷) were used for labeling. 
2. U2OS cells were incubated with 500 μM biotin tyramide containing medium for 30 min.
3. 1 mM hydrogen peroxide was added to the medium to activate APEX reaction for 1 min, followed by immediate quenching of reaction with ice-cold quenching buffer (1xPBS, 10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox).
4. After washing with cold quenching buffer for three times, the cells were collected from plates with scrapers.
5. Cells were lysed in lysis buffer (100 mM NaPO4, PH 8.0, 8 M Urea, 0.05% SDS, 10 mM sodium azide, 10 mM sodium ascorbate, 5 mM Trolox supplemented with 500 unit benzonase to remove DNA and RNA) and rotated at RT for 15 min. Then SDS was increased to 1% and sonication was performed in a water bath sonicator for 10 min. 
6. Protein concentration was measured using 2-D quant kit following manufacturer’s instructions. The denatured proteins were reduced with 5 mM TCEP and alkylated with 10 mM Iodoacetamide.
7. Equal amount of proteins (0.5 – 1 mg) from light and heavy group were mixed together and then the mixed cell lysates were diluted with equal volume of ddH₂O to reduce the concentration of urea to 4 M and SDS to 0.5%.
8. The samples were incubated with streptavidin magnetic beads (100 μL) at 4°C overnight.
9. After four washes with wash buffer (100 mM NaPO₄, pH 8.0, 4 M urea), the beads were resuspended in 100 mM TEAB, 2 M Urea supplemented with 10 ng/μl Trypsin, 5 ng/μl Lys-C for digestion overnight. 

    10. The digested products were collected and added with 1% formic acid (final concentration) for LC-MS/MS analysis.