Modified affinity separation strategy

a) Functional pull-down: HL-60 cells were grown in a T-75 corning flask in 20 mL media until the concentration reached 1 x 106 cells/mL. The cells were incubated under 37 oC with functional PTL probe (7, 40 μM, the probe was pre-dissolved in DMSO to form a 10 mM stock solution) for 8 hrs and then lysed by RIPA buffer. The protein concentration of the lysates was determined by BCA assay. In most cases, the

concentration is between 1.1 to 1.3 mg/mL. The concentration was adjusted to 1 mg/mL by adding extra RIPA buffer.

To functional labeled HL-60 cell lysate (1 mg/mL, 0.5 mL) was added Biotin-N3

probe (28, 10 mM in DMSO, 3.3 μL), ligand (10 mM in DMSO, 3.8 μL), sodium ascorbate (50 mM in H2O, 7 μL) and CuSO4 (50 mM in H2O, 7 μL) subsequently. The

mixture was allowed to gently agitate at room temperature for 2.5 hr before loading to a monomeric avidin agrose column. The column contains 2 mL agrose and was washed with blocking buffer (10 mL, 2 mM D-biotin in PBS), regeneration buffer (12 mL, 0.1 M glycine, pH2.8) and amine free PBS buffer (12 mL, generated from pierce BupH™ Phosphate Buffered Saline pack) before using. After loaded, the protein sample was incubated the monomeric avidin column for 0.5 hr at room temperature and then, the column was washed with amine free PBS buffer (12 mL) to remove non-bound protein, blocking buffer (12 mL) to elute labeled protein and regeneration buffer (12 mL) to elute and regenerate the resin for further use. The fractions from both the blocking buffer and regeneration buffer were collected to a 15 mL centrifugal filter unit (Millipore, amicon ultra 15, PL membrane, 3 KDa) and neutralized with TrisHCl buffer (1 mL, pH 9.5). The filter unit was centrifuged at 4000 x g for 45 min to centrifuge the sample to about 300 μL. An aliquot of the final protein sample was analyzed by SDS-PAGE (Invitrogen NuPAGE Bis-Tris minigel, 1 mm, 10 wells, 4%-12%, 185 V, 35 min), followed by silver staining. The protein sample was precipitated by 10% TCA (procedure see below) to remove salt and analyzed by mass spectrometry (at Taplin Mass Spectrometry Facility, Harvard Medical School).

b) Non-functional pull-down: HL-60 cells were grown in a T-75 corning flask in 20 mL media until the concentration reached 1 x 106 cells/mL. The cells were incubated under 37 oC with non-functional PTL probe (8, 40 μM, the probe was pre-dissolved in DMSO to form a 10 mM stock solution) for 8 hrs and lysed by RIPA buffer. The protein concentration was determined by BCA assay (Pierce BCA protein assay kit). In most cases, the concentration is between 1.4 to 1.6 mg/mL. The concentration was adjusted to 1 mg/mL by adding extra RIPA buffer.

To non-functional labeled HL-60 cell lysate (1 mg/mL, 0.5 mL) was added Biotin-N3 probe (15, 10 mM in DMSO, 3.3 μL), ligand (10 mM in DMSO, 3.8 μL),

sodium ascorbate (50 mM in H2O, 7 μL) and CuSO4 (50 mM in H2O, 7 μL)

subsequently. The mixture was allowed to gently agitate at room temperature for 2.5 hr before loading to a monomeric avidin agrose column. The column separation process was the same as described above. An aliquot of the protein sample was analyzed by SDS-PAGE (Invitrogen NuPAGE Bis-Tris minigel, 1 mm, 10 wells, 4%-12%, 185 V, 35 min), followed by silver staining. The protein sample was precipitated by 10% TCA to remove salt and analyzed by mass spectrometry (at Taplin Mass Spectrometry Facility, Harvard Medical School).

c) Competitive pull-down experiment: HL-60 cells were grown in a T-75 corning flask in 20 mL media until the concentration reached 1 x 106 cells/mL. The cells were firstly incubated under 37 oC with PTL (20 μM, the probe was pre-dissolved in DMSO to form a 10 mM stock solution) for 4 hrs and then, with functional PTL probe (7, 20 μM) for another 3 hrs. Cells were lysed by RIPA buffer and protein concentration was

determined by BCA assay (Pierce BCA protein assay kit). In most cases, the concentration is between 1.1 to 1.3 mg/mL. The concentration was adjusted to 1 mg/mL by adding extra RIPA.

To competitively labeled HL-60 cell lysate (1 mg/mL, 0.5 mL) was added Biotin-N3 probe (15, 10 mM in DMSO, 3.3 μL), ligand (10 mM in DMSO, 3.8 μL),

sodium ascorbate (50 mM in H2O, 7 μL) and CuSO4 (50 mM in H2O, 7 μL)

subsequentially. The mixture was allowed to gently agitate at room temperature for 2.5 hrs before loading to a monomeric avidin agrose column. The column separation process was the same as described above. An aliquot of the protein sample was analyzed by SDS-PAGE (Invitrogen NuPAGE Bis-Tris minigel, 1 mm, 10 wells, 4%-12%, 185 V, 35 min), followed by silver staining. The protein sample was precipitated by 10% TCA to remove salt and analyzed by mass spectrometry (at Taplin Mass Spectrometry Facility, Harvard Medical School).

d) TCA (Trichloroacetic acid) protein precipitation: After concentration by filter units, the protein samples were diluted to 400 μL using cold deionized H2O. After the

addition of 100 μL 100% TCA solution (prepared by dissolving 10 g TCA to a total volume of 10 mL in deionized H2O), the protein samples were incubated on ice until a

white cloudy precipitation was observed (about 20 min). The samples were centrifuged at 14000 x g at 4 C for 20 min to yield the protein pellets. After careful removal TCA solution, 200 μL cold acetone was added to each protein pellet. The samples were votexed and centrifuged again for 10 min at 14000 x g at 4 C and the acetone was

removed with precaution. At last, the residual acetone was dried in speed-vac over night to yield the final protein pellets that were ready for mass spectrum analysis.

e) Mass spectrometry analysis: the above mentioned protein samples were analyzed at Taplin Mass Spectrometry Facility, Harvard Medical School (https://taplin.med.harvard.edu/intro). Briefly, proteins in each sample were digested by trypsin and the resulting peptide fragments were analyzed and sequenced by microcapillary LC/MS/MS techniques. In order to identify proteins from a sample, the peptides information obtained from the above experiment was then compared to IPI databases (International Protein Index). Any protein with three or more unique peptide matches compared to a known protein is considered confidently identified. Proteins with less than three matches are not considered confidently identified without further inspection of the data.

2.6 NMR Spectrum