Supplementary Figure 1 Method development. Titration experiments to determine standard antibody:lysate concentration. Lysates (~2 mg of total proteins) were prepared from cells expressing FLAG- tagged CBX2 (a) or SCMH1 (b) and either anti-flag or a number of recombinant antibodies were titred in for immunoprecipitation. Shown is a Western blot of the Flag-tagged antigen. The left of each Panel, labeled IP, indicates the proteins captured in the IP and on the right, labeled FT, are the proteins remaining in the lysate after immunoprecipitation. Most of the recombinant Fabs approached saturation when 2 µg of antibody or 10 µl of anti-flag beads were used per 2 mg of lysate. Nature Methods doi:10.1038/nmeth.3472
Supplementary Figure 2 Determination of a suitable cutoff for the elimination of background contaminants. (a) The graph shows the number of preys recovered at a given percentage of occurence among all Fab or IgG purifications. From this graph it seems that 8% is an inflection point indicating a good cut-off frequency. Below 8%, increase of percentage causes rapid drop in the number of preys recovered while above 8% the drop in frequency slows down dramatically. This implies that preys appearing in less than 8% purifications are more likely to be specific. Those appearing in more than 8% purification are more likely to be non-specific (regardless of the occurrence, the number of preys is similar). (b) Venn diagrams showing overlap between proteins identified in immunoprecipitations using >1,000 different recombinant antibodies and those from
Crapome repository 11. On the left, we compared all the proteins detected in all immunoprecipitations and on the right only those proteins that appear in more than 8% of all purifications.
Supplementary Figure 3 Comparison between IP-WB and IP-MS methods. Fabs or IgGs (phagemid IDs are indicated) against several targets were used to immunoprecipitate their antigens from HEK293 cell extracts expressing their respective Flag-tagged proteins. The immunoprecipitated proteins were then detected using either WB or MS. The spectral counts from IP-MS experiments are indicated below each panel.
Supplementary Figure 4 Comparison of staining patterns. Flag-tagged cell lines were stained using either anti-flag antibody or Fabs against 5 different proteins, listed on the right, and the staining pattern was compared. For these Fabs, the localization patterns are very similar to the anti-flag staining. DAPI staining was used to highlight the nuclei. Flag-tagged WDR5, shows nucleolar staining, but the anti-wdr5 Fab does not; the nucleolar staining is likely due to the over-expression of Flagtagged WDR5. Scale represents 20 µm.
Supplementary Protocol: IP- MS NOTE: Everything on ice or at 4 o C unless indicated otherwise. All incubations are with rotation. NOTE: Use non-autoclaved tubes and tips! A. Preparing cell lysate (HEK293 cells) 1. Thaw frozen cell pellets immediately with 1X High Salt AFC buffer (1 ml per 15 cm plate) with protease and phosphatase inhibitors 2. Perform 3 freeze- thaw cycles by moving samples between ethanol/dry ice and 37 C water bath, mixing frequently to prevent temperature from reaching above 4 C 3. Sonicate, (Branson sonicator) 5x (0.3s on/0.7 s off) per 1 ml, 8x per 2 ml and 12x per 5 ml 4. Incubate for 30min at 4 C with benzonase nuclease to remove RNA and DNA (to the final concentration of 12.5-25 units/ml). 5. Centrifuge at 13,000 rpm for 30min at 4 C. Discard pellet and retain supernatant. 6. Measure protein concentration (nanodrop), usually 7-10 mg/ml B. Antibody binding 1. Use 2-5 mg of total protein per IP. Add ~2 μg of antibody into the lysate and incubate at 4 o C overnight (depending on the stability of the proteins), 2 hrs incubation is sufficient for most antibodies. 2. Wash 20 µl of magnetic beads per IP twice with 100 µl high salt AFC buffer. After last wash resuspend in 20 µl of low salt AFC buffer and add 20 µl to the lysates with antibodies. 3. Incubate for 2-4 hrs at 4 o C. 4. Wash the beads with 1 ml of 1x low salt lysis buffer 3x at 4 o C for 5-10 min and 2x with low salt lysis buffer without detergent. 5. Elute 4x50 µl of 0.5 M ammonium hydroxide, combine the elutions and flash freeze in liquid nitrogen 6. Dry and perform trypsin digest according to protocol below. C. Trypsin Digestion NOTE: All the steps should be done in HPLC grade water or 50mM NH4HCO3 NOTE: Use non- autoclaved tubes and tips NOTE: Make sure you work in a clean environment to prevent keratins and polymer contamination, hood is not necessary but careful work environment is essential! 1. Dry NH4OH samples in the speed vac (about 1 1.5 hrs) 2. Reconstitute in 44 µl of 50 mm NH4HCO3 3. Add 1 µl of 100mM TCEP- HCL. Shake at 37 o C for 1 hr
4. Cool to room temperature and add 1 µl 500 mm iodoacetamide. Shake at room temperature and in dark for 45 mins 5. Add 1 µg Trypsin. Shake overnight at 37 o C 6. Add 2µl acetic acid to stop the reaction 7. Store at 4 o C 8. The volume is 50.5 µl D. Desalting (for Zip- Tip) NOTE: The maximum volume for Zip Tip is 10 µl 1. Equilibration: Aspirate 10µl Wetting and Equilibration solution into zip- tip. Dispense waste. Repeat twice. Aspirate 10µl with washing solution. Dispense waste. Repeat twice. 2. Binding and washing: Aspirate 10υl and dispense into the sample, repeat at least 20 times so all the peptides are bound to the tip (do not throw away the samples just pipette up and down). Aspirate 10υl of washing solution twice and dispense. 3. Elution: Dispense 10υl of Wetting and Equilibrating solution into 96 well plate four times (Final sample volume is 40 µl). 4. Dry the samples. 2. AFC (High Salt) (Lysis buffer) Final con Stock For 40 ml 10X buffer Tirs- HCl, ph7.9 10mM 2 M 2 ml NaCl 420mM 5 M 33.6 ml NP- 40 0.1% 10% 4 ml H2O 0.4 ml Add protease inhibitor just before use Add 1mM Ni to the buffer for demethylases 3. AFC (Low Salt) (Dialysis/Wash buffer) Final con Stock For 40 ml 10X buffer Tirs- HCl, ph7.9 10 mm 2 M 2 ml NaCl 100 mm 5 M 8 ml NP- 40 0.1% 10% 4 ml H2O 26 ml Add protease inhibitors just before use, same as high salt 4. Wetting and Equilibration solution 70% CAN in 0.1% FA
5. Washing solution 100% H20 in 0.1% FA 6.Catalogue # Zip Tip catalogue # ZTC18M960 (Millipore) Mass Spec Plate Cat no: PCR- 96- FS- C (Axygen scientific from VWR)
Supplementary Note Trypsin Digestion Protocols Lab 1 (Greenblatt Lab): Samples were reconstituted in 44 µl of 50 mm NH4HCO3, and 1 µl of 100mM TCEP-HCL (Thermo Fisher) was added. Samples were incubated at 37 C for 1 hr with shaking and then cooled to room temperature. 1 µl 500 mm iodoacetamide was added and samples incubated in the dark at room temperature for 45 mins, following which 1 µg of trypsin (Promega, Catalog #V5113) was added per sample and samples incubated overnight at 37 C. The next day, 2µl of acetic acid was added to stop the reaction. The final volume was 50.5µl. Desalting was performed using ZIP-TIP (Millipore, Catalog #ZTC18M960). ZIP-TIP were equilibrated by aspirating and dispensing 10 µl of wetting and equilibration solution two times followed by 2x10µl of washing solution. Binding was performed by aspirating and dispensing 10 µl of sample at least 20x to allow all the peptides to bind to the tip. The tips were then washed 2x with 10µl of washing solution, and the bound peptides eluted into a 96 well plate with 4x10µl of wetting and equilibrating solution (Axygen, Catalogue #PCR-96-FS-C). After drying and acidification to 1% formic acid, the peptides were ready to be injected into LC- MS/MS. Lab 2 (Gingras lab): Trypsin digestion was carried out on the beads. Beads with bound proteins were subjected to one rapid wash in 0.5 ml lysis buffer followed by on-bead trypsin hydrolysis for 4 h at 37 C with 0.25 µg trypsin (Sigma, Trypsin Singles). Released peptides in solution were removed and further digested overnight with an additional 0.25 µg of trypsin at 37 C. 2µl of acetic acid was added to stop the reaction. The sample was desalted using ZIP-TIP as described above. Lab 3 (Aebersold/Gstaiger): Samples were reconstituted in Resuspend in 50µl 8M urea 50 mm ammonium bicarbonate, reduced with 5 mm TCEP for 30 min and alkylated with10 mm iodoacetamide for 30 min. Samples were diluted to 300µl with 50 mm ammonium bicarbonate to reduce urea concentration and digested overnight with 1ug
trypsin. The next morning samples were acidified to 1.25 % with formic acid and solid phase extraction as per manufacturer using C18 ultramicrospin columns (Nest Group part SS18V) was carried out. Samples were resuspended 20 µl (0.1% formic acid 2% ACN). Lab 4 (Paulovich lab): Samples were alkylated with 50mM iodoacetamide at room temperature in the dark for 30 minutes, diluted 10 in 10 mm Tris, ph 8, and digested with Trypsin Gold (Promega, Madison, WI) at a protein-to enzyme ratio of 50:1 overnight at 37 C. The reaction was quenched by adding formic acid to a final concentration of 1%. Samples were desalted with Discovery SPERP reverse phase C18 columns(supelco) according to manufacturers protocols. Briefly, the columns were rinsed thrice with 80% acetonitrile / 0.1% formic acid (FA), equilibrated four times with 0.1% FA. The sample was applied to the column, washed 4 with 0.1% FA, and eluted with 2 washes of 50% acetonitrile / 0.1% FA, followed by 2 washes of 80% acetonitrile / 0.1% FA. The sample was concentrated by vacuum centrifugation to 20 µl. Lab 5 (Thermo): Samples were reconstituted with 50µl of 4M Urea, 300mM Tris, 2.5% n-propanol and 10mM DTT, vortexed for 10 min and incubated for 30 minutes at 37C. 4µl of 500mM IAA (iodoacetic acid) in 1M NH4HCO3 was added to the final concentration of 40 mm per well. The samples were incubated at room temperature in complete darkness for 30 min. 20 µg trypsin was with 500µl 25mM Acetic Acid (100µg vial w/2500µl AA) and incubated at 37C for 5 minutes. 75µl of 50mM Tris, 5mM CaCl2 ph 8.0 was added directly to each well followed by addition of 25ul Trypsin/AA to each well. The samples were incubated at 37C for 20 hours. Samples were quenched with 10 µl of Quench mix (20µl PRTC (Pierce Peptide Retention Time Cal Mixture 5uM), 200µl 100%Formic Acid, 16µl Glucagon [1mg/ml in H2O], 764µl LCM grade H2O.