the data presented here uses rapid immunoprecipitation mass spectrometry of endogenous protein ( rime )   to interrogate the interactomes of the estrogen receptor ( er ) and progesterone receptor ( pr ) under either estrogenic conditions ( complete media ) or with additional progesterone treatment . this proteomics study describes the effect of activating a single nuclear receptor or two receptors with their respective ligands in breast cancer cell lines and is part of a study that identifies the synergistic roles of er and pr in breast cancer . all experiments were performed in the breast cancer cell lines mcf-7 and t47-d , both of which express sufficient levels of er and pr . the er+/pr+ cell lines , mcf-7 and t47-d cells were grown in silac dmem ( paa ; e15 - 086 ) supplemented with 10% dialysed serum ( sigma - aldrich ; f0392 ) and 800  mm l - lysine c6n2 hydrochloride and 482  mm l - arginine c6 n4 hydrochloride ( sigma - aldrich ) for  heavy-labelled media or 800  mm l - lysine c6n2 hydrochloride and 482  mm l - arginine c6n4 hydrochloride for  light-labelled media . alternative silac dmem compatible with mcf-7 and t47-d cells also yield similar results ( thermo ; 89985 ) . cells switching to silac media were also supplemented with additional glutamate . to avoid clonal effects , cells are expanded in silac media and harvested immediately after an appropriate level of label incorporation is achieved , thus heavy or light labeled cells were not maintained long term . culture cells were supplemented with 100  nm of progesterone or 10  nm r5020 for 4  h. the ligands were diluted in dmem ( no serum ) and all dilutions and stock solutions were maintained in glass vials . cells were grown in 15  cm culture ( nunc ) dishes and harvested at ~80% confluency . cells were crosslinked by replacing media with fresh pre - warmed dmem ( 10  ml , without fbs ) containing 1% formaldehyde ( tebu bioscience ; 18814 ) . after 8  min of crosslinking , 1  ml of 2.5  m glycine was added to each plate ( final volume 11  ml ) . plates of cells were then washed twice with cold pbs before the cells were harvested in 1  ml of pbs using a cell scraper . cells were then frozen at 80  c for storage and subsequent steps were performed at a later date . 20  g of er ( santa cruz ; sc-543 , lot - a2213 ) or pr ( santa cruz ; sc-7208 , lot h2312 ) antibodies conjugated to 100  l of magnetic protein a beads were used for each rime experiment . to prepare the conjugate , 100  l of beads were washed three times with 1  ml of 0.5% bsa in pbs . the beads were then resuspended in 100  l of this buffer and 20  gs of antibody was added . the mixture was topped up with additional bsa / pbs buffer ( 250  l ) and then incubated overnight at 4  c ( rotating ) . the beads were then washed again 3 times in bsa / pbs to remove any unconjugated antibody and then resuspended in 100  l of bsa / pbs buffer . this final antibody - bead conjugate is then subsequently added to the cell lysate for immunoprecipitation . for each rime experiment , approximately 20 million cells each ( equivalent to 215  cm dish ) of treatment and control grown in heavy or light silac conditions were mixed . the cells are then resuspended in 10  ml of lb1 buffer ( 50  mm hepes - koh [ ph 7.5 ] , 140  mm nacl , 1  mm edta , 10% glycerol , 0.5% np-40 or igepal ca-630 , and 0.25% triton x-100 ) and incubated at 4  c for 10  min ( rotating ) . the cells were then pelleted at 2000  g for 5  mins ( 4  c ) and the supernatant removed . the resulting pellet was then resuspended in 10  ml of lb2 buffer ( 10  mm tris hcl [ ph 8.0 ] , 200  mm nacl , 1  mm edta , and 0.5  mm egta ) and incubated again at 4  c for 5  min ( rotating ) . this was then centrifuged at 2000  g for 5  mins and the supernatant was removed . hcl [ ph 8 ] , 100  mm nacl , 1  mm edta , 0.5  mm egta , 0.1% na - deoxycholate , and 0.5% n - lauroylsarcosine ) . 1020 million cells ) were resuspended in 300  l of lb3 and transferred to a 1.5  ml eppendorf tube and sonicated in a bioruptor sonicator for 15  min ( 30  s on/30  s off , high setting ) . the sonicated lysates for each plate were then pooled and 10% volume of 10%triton - x solution was added . this mixture was then centrifuged at 12,000  g for 10  min to pellet cell debris . the supernatant was carefully removed and 100  l of beads pre - incubated with antibody was added . subsequently , the lysate - bead mixture was then placed on a magnetic rack and the supernatant removed . the resulting beads were then washed 10 times in ripa buffer ( 50  mm hepes ph 7.6 , 1  mm edta , 0.7% na deoxycholate , 1% np-40 , 0.5  m licl ) and then twice in 1  ml of 100  mm ammonium hydrogen carbonate ( freshly prepared ) . after the last wash , the beads were briefly spun down and any residual liquid was removed . beads were then either frozen ( 20  c ) or directly processed for peptide digestion . bead bound protein complexes were then digested by adding 100  ng of trypsin in 10  l of 100  mm nh4hco3 onto the beads . the next day , an additional 10  l of trypsin was added , vortexed and incubated for an additional 4  h. the supernatant was then removed and transferred to a glass vial containing 1  l of formic acid ( 5% final concentration ) . the acidified samples were then bound to a c18 ultra - micro spin column ( harvard apparatus ) and washed using a 0.1% solution of formic acid . the samples were then re - suspended in 0.1% formic acid and loaded on - to an ltq orbitrap velos ( thermo ) mass spectrometer and then run using settings previously described . the acquired ms spectra were then analysed using proteome discoverer v1.3 ( thermo scientific ) in combination with the mascot search engine ( matrix science ) against the swissprot human database . the search parameters allowed for one missed cleavage and a precursor ion search tolerance of 10  ppm with 0.6  da for ms / ms ions . additionally , the variable modifications oxidized methionine and de - amidated asparagine and glutamate residues were included as search parameters . the appropriate heavy amino acid silac settings were set as fixed modifications . a false discovery rate ( fdr ) was calculated using a reversed decoy database with a target of less than 1% . for each rime experiment , only proteins identified in both cell lines were considered ( fig . the er+/pr+ cell lines , mcf-7 and t47-d cells were grown in silac dmem ( paa ; e15 - 086 ) supplemented with 10% dialysed serum ( sigma - aldrich ; f0392 ) and 800  mm l - lysine c6n2 hydrochloride and 482  mm l - arginine c6 n4 hydrochloride ( sigma - aldrich ) for  heavy-labelled media or 800  mm l - lysine c6n2 hydrochloride and 482  mm l - arginine c6n4 hydrochloride for  light-labelled media . alternative silac dmem compatible with mcf-7 and t47-d cells also yield similar results ( thermo ; 89985 ) . cells switching to silac media were also supplemented with additional glutamate . to avoid clonal effects , cells are expanded in silac media and harvested immediately after an appropriate level of label incorporation is achieved , thus heavy or light labeled cells were not maintained long term . culture cells were supplemented with 100  nm of progesterone or 10  nm r5020 for 4  h. the ligands were diluted in dmem ( no serum ) and all dilutions and stock solutions were maintained in glass vials . cells were grown in 15  cm culture ( nunc ) dishes and harvested at ~80% confluency . cells were crosslinked by replacing media with fresh pre - warmed dmem ( 10  ml , without fbs ) containing 1% formaldehyde ( tebu bioscience ; 18814 ) . after 8  min of crosslinking , 1  ml of 2.5  m glycine was added to each plate ( final volume 11  ml ) . plates of cells were then washed twice with cold pbs before the cells were harvested in 1  ml of pbs using a cell scraper . cells were then frozen at 80  c for storage and subsequent steps were performed at a later date . 20  g of er ( santa cruz ; sc-543 , lot - a2213 ) or pr ( santa cruz ; sc-7208 , lot h2312 ) antibodies conjugated to 100  l of magnetic protein a beads were used for each rime experiment . to prepare the conjugate , 100  l of beads were washed three times with 1  ml of 0.5% bsa in pbs . the beads were then resuspended in 100  l of this buffer and 20  gs of antibody was added . the mixture was topped up with additional bsa / pbs buffer ( 250  l ) and then incubated overnight at 4  c ( rotating ) . the beads were then washed again 3 times in bsa / pbs to remove any unconjugated antibody and then resuspended in 100  l of bsa / pbs buffer . this final antibody - bead conjugate is then subsequently added to the cell lysate for immunoprecipitation . for each rime experiment , approximately 20 million cells each ( equivalent to 215  cm dish ) of treatment and control grown in heavy or light silac conditions were mixed . the cells are then resuspended in 10  ml of lb1 buffer ( 50  mm hepes - koh [ ph 7.5 ] , 140  mm nacl , 1  mm edta , 10% glycerol , 0.5% np-40 or igepal ca-630 , and 0.25% triton x-100 ) and incubated at 4  c for 10  min ( rotating ) . the cells were then pelleted at 2000  g for 5  mins ( 4  c ) and the supernatant removed . the resulting pellet was then resuspended in 10  ml of lb2 buffer ( 10  mm tris hcl [ ph 8.0 ] , 200  mm nacl , 1  mm edta , and 0.5  mm egta ) and incubated again at 4  c for 5  min ( rotating ) . this was then centrifuged at 2000  g for 5  mins and the supernatant was removed . hcl [ ph 8 ] , 100  mm nacl , 1  mm edta , 0.5  mm egta , 0.1% na - deoxycholate , and 0.5% n - lauroylsarcosine ) . 1020 million cells ) were resuspended in 300  l of lb3 and transferred to a 1.5  ml eppendorf tube and sonicated in a bioruptor sonicator for 15  min ( 30  s on/30  s off , high setting ) . the sonicated lysates for each plate were then pooled and 10% volume of 10%triton - x solution was added . this mixture was then centrifuged at 12,000  g for 10  min to pellet cell debris . the supernatant was carefully removed and 100  l of beads pre - incubated with antibody was added . subsequently , the lysate - bead mixture was then placed on a magnetic rack and the supernatant removed . the resulting beads were then washed 10 times in ripa buffer ( 50  mm hepes ph 7.6 , 1  mm edta , 0.7% na deoxycholate , 1% np-40 , 0.5  m licl ) and then twice in 1  ml of 100  mm ammonium hydrogen carbonate ( freshly prepared ) . after the last wash , the beads were briefly spun down and any residual liquid was removed . beads were then either frozen ( 20  c ) or directly processed for peptide digestion . bead bound protein complexes were then digested by adding 100  ng of trypsin in 10  l of 100  mm nh4hco3 onto the beads . the next day , an additional 10  l of trypsin was added , vortexed and incubated for an additional 4  h. the supernatant was then removed and transferred to a glass vial containing 1  l of formic acid ( 5% final concentration ) . the acidified samples were then bound to a c18 ultra - micro spin column ( harvard apparatus ) and washed using a 0.1% solution of formic acid . the samples were then re - suspended in 0.1% formic acid and loaded on - to an ltq orbitrap velos ( thermo ) mass spectrometer and then run using settings previously described . the acquired ms spectra were then analysed using proteome discoverer v1.3 ( thermo scientific ) in combination with the mascot search engine ( matrix science ) against the swissprot human database . the search parameters allowed for one missed cleavage and a precursor ion search tolerance of 10  ppm with 0.6  da for ms / ms ions . additionally , the variable modifications oxidized methionine and de - amidated asparagine and glutamate residues were included as search parameters . the appropriate heavy amino acid silac settings were set as fixed modifications . a false discovery rate ( fdr ) was calculated using a reversed decoy database with a target of less than 1% . for each rime experiment , only proteins identified in both cell lines were considered ( fig .
nuclear receptors play an important role in transcriptional regulation of diverse cellular processes and is also relevant in diseases such as cancer . in breast cancer , the nuclear receptors 
 estrogen receptor ( er ) and progesterone receptor ( pr ) are classical markers of the disease and are used to classify breast cancer subtypes . using a recently developed affinity purification ms technique ( rime ) [ 1 ] , we investigate the protein interactors of er and pr in breast cancer cell lines upon stimulation by the ligands  estrogen and progesterone . the data is deposited at proteomexchange ( pxd002104 ) and is part of a publication [ 2 ] that explains the link between the two nuclear receptors and potential consequences of this in breast cancer . in this manuscript , we describe the methodology used and provide details on experimental procedures , analysis methods and analysis of raw data . the purpose of this article is to enable reproducibility of the data and provide technical recommendations on performing rime in hormonal contexts .