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\starttext
  Remarkably, our comparative analysis of Gcn5p purifications yielded strong candidates for six new Gcn5p interactors. YCR082W, a nonessential gene product (Winzeler et al. 1999; Giavert et al. 2002) with unknown function, was found in all five Gcn5p purifications but was not recovered with any of the other baits that we analyzed. YCR082W exhibits a two--hybrid interaction with Ahc1p (Uetz et al. 2000; Ito et al. 2001), which together with Gcn5p is a member of the ADA histone acetyltransferase complex (Eberharter et al. 1999). Another candidate is Msn4p, a nonessential (Estruch et al. 1993; Winzeler et al. 1999) major transcriptional regulator of stress responses (Treger et al. 1998). Msn4p was recovered in four of the five Gcn5p pull down experiments but was not recovered with any of the other baits. This finding is interesting in the light of evidence that promoters activated by Msn4p and its partner Msn2p show increased histone H4 acetylation (Deckert et al. 2001). Other potential interaction partners include YPL047W (present in two of the HPM purifications and the TAP purification), histones Hta1p/Hta2p and Imd4p (in TAP, modified TAP and one HPM pulldown). Other gene products recovered in more than two of the experiments are mostly ribosomal proteins that are likely contaminants. Finally, the interaction observed between Gcn5p and Swi1p in the TAP tag experiment was previously proposed only on the basis of their synthetically lethal genetic interaction (Pollard et al 1997).\par
  \subsection{Screening for Interactions}
    Having established the relative reproducibility of TAP--MudPIT and the comparability of the HPM tag to other available bipartite affinity tags, we set out to address three issues. First, we wished to determine what fraction of TAP--MudPIT experiments yield usable results. Second, we hoped to determine whether the parallel application of MudPIT to numerous baits would enable us to cull nonspecific contaminants by comparing protein identifications across multiple experiments. Third, we wanted to test whether it will be feasible for an investigator in a cell biology laboratory to work at the scale needed to dissect a biological pathway or process by systematic application of MudPIT to a few dozen gene products. To address these questions, we screened for new protein--protein interactions in a test set of 25 gene products involved in transcription and progression through mitosis. Table x.x summarizes the results and gives an overview of potential new interactors. The complete data set may be found in the supporting online material.\par
  \splitfloat[lines=8]
    {
      \placetable
        [top]
        {
          \select{caption}
            {Potential New Interactors for a Test Set of HPM Tagged Proteins.}
            {{\bf Potential New Interactors for a Test Set of HPM Tagged Proteins.} Samples were prepared and analyzed as described in Graumann et al (2004). Column ``Known interactors---Total'' lists the number of physical/genetic interactions reported for the bait in the combined GRID/MIPS/YPD databases. ``Known interactors---Recovered'' represents the number of known physical/genetic interactors experimentally retrieved in this study. Partners marked ``\,*\,'' are reported to interact physically as well as genetically. Column ``Potential new interactors'' contains all gene products identified by TAP--MudPIT, which are not listed as known interactors and are recovered in association with less than 20\% of the baits analyzed (n=22).}
        }
    }
    {
      \setupTABLE[row][1][topframe=on]
      \setupTABLE[row][last][bottomframe=on]
      \bTABLE[frame=off,split=repeat,splitmethod=b,style={\switchtobodyfont[small]\setupinterlinespace[line=2.8ex]}]
        \bTABLEhead
          \bTR
            \bTD[nr=3,nc=2,bottomframe=on] Bait\eTD
            \bTD[nc=5,bottomframe=on] Known interactors\eTD
            \bTD\eTD
            \bTD[nr=3,bottomframe=on] Potential new interactors\eTD
            \eTR\bTR
            \bTD[bottomframe=on] Total\eTD
            \bTD\eTD
            \bTD[nc=3,bottomframe=on] Recovered\eTD
          \eTR
          \bTR[bottomframe=on]
            \bTD phys./gen.\eTD
            \bTD[width=0.5em]\eTD
            \bTD phys.\eTD
            \bTD[width=0.5em]\eTD
            \bTD genet.\eTD
            \bTD[width=0.5em]\eTD
          \eTR
        \eTABLEhead
        \bTABLEbody
          \bTR
            \bTD Bim1p--HPM\eTD
            \bTD\eTD
            \bTD 6/57\eTD
            \bTD\eTD
            \bTD 1\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Rpb2p, Rpl12A/Bp, Rpl22Ap, Rps25A/Bp, Rps29Ap, Rps5p, YGR161C--Cp\eTD
            \eTR\bTR
            \bTD Cdc20p--HPM\eTD
            \bTD\eTD
            \bTD12/3 \eTD
            \bTD\eTD
            \bTD 6\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Bub3p, Cct4p, Cct6p, Cct7p, Cct8p, Hef3p, Ilv6p, Pnc1p, Rfa1p\eTD
          \eTR
          \bTR
            \bTD Chk1p--HPM\eTD
            \bTD\eTD
            \bTD 16/0\eTD
            \bTD\eTD
            \bTD---\eTD
            \bTD\eTD
            \bTD---\eTD
            \bTD\eTD
            \bTD Act1p, Car2p, Gpd2p, Hht1p, Hht2p, Htb2p, Htb1p, Htz1p, Pnc1p\eTD
          \eTR
          \bTR
            \bTD Cla4p--HPM\eTD
            \bTD\eTD
            \bTD 15/77\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Rpl17Bp,\crlf Rpl17Ap,\crlf Rpl19Bp,\crlf Rpl19Ap\eTD
            \bTD\eTD
            \bTD Pbp1p, Pre8p, Rpl36Ap, Rpl36Bp, Rpl7Ap, Rpl7Bp, Rpp2Ap, Rps2p, Sec23p, Skm1p, YBR225Wp, Yhb1p\eTD
          \eTR
          \bTR[bottomframe=on]
            \bTD Dbf2p--HPM\eTD
            \bTD\eTD
            \bTD 27/9\eTD
            \bTD\eTD
            \bTD 3\eTD
            \bTD\eTD
            \bTD Dbf20p,\crlf Mob1p*\eTD
            \bTD\eTD
            \bTD Adh5p, Caf20p, Car2p, Cdc33p, Emi2p, Gfa1p, Gly1p, Gpd2p, Hsp42p, Ilv6p, Pnc1p, Pro1p, Rib4p, Sec23p, Shm2p, Snf1p, Trp3p, Tub2p\eTD
          \eTR
          \bTR[topframe=on]
            \bTD Gcn5p--HPM\eTD
            \bTD\eTD
            \bTD 99/12\eTD
            \bTD\eTD
            \bTD 18\eTD
            \bTD\eTD
            \bTD Ngg1p*\eTD
            \bTD\eTD
            \bTD Ade3p, Eft2p, Eft1p, Gfa1p, Glc7p, Msn4p, Ppz2p, Rpl16Ap, Rpp2Ap, Rpp2Bp, Rps25Ap, Rps25Bp, Rps29Bp, Sds22p, Sod2p, Tfc1p, Trp3p, Tub2p, Ura7p, YCR082Wp, Yhb1p, Ypi1p, YPL047Wp, YPL137Cp, Ysh1p\eTD
          \eTR
          \bTR
            \bTD Glc7p--HPM\eTD
            \bTD\eTD
            \bTD 177/9\eTD
            \bTD\eTD
            \bTD 28\eTD
            \bTD\eTD
            \bTD Ppz2p*,\crlf Ppz1p*,<br/>Reg1p*\eTD
            \bTD\eTD
            \bTD Abf1p, Ade16p, Ade17p, Ahp1p, Bmh1p, Bmh2p, Ccr4p, Cka2p, Eno1p, Fun21p, Gal83p, Hsp60p, Imp2p, Mor1p,   Pdc1p, Pgk1p, Pol2p, Rpp2Ap, Snf1p, Sol1p, Sol2p, YBR225Wp, YDR474Cp, YER158Cp, YGR237Cp, YHR097Cp, YPL137Cp\eTD
          \eTR
          \bTR[bottomframe=on]
            \bTD Ino4p--HPM\eTD
            \bTD\eTD
            \bTD 52/0\eTD
            \bTD\eTD
            \bTD 1\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Act1p, Mdn1p, Pmd1p, Xrs2p\eTD
          \eTR
          \bTR[topframe=on]
            \bTD Lte1p--HPM\eTD
            \bTD\eTD
            \bTD 48/12\eTD
            \bTD\eTD
            \bTD 5\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Ade4p, Aro2p, Asc1p, Asn1p, Bcy1p, Bmh1p, Caf20p, Car2p, Cdc33p, Eft2p, Eft1p, Emi2p, Eno1p, Eno2p, Flo8p, Gad1p, Glk1p, Glt1p, Gly1p, Gpm1p, Gua1p, Hef3p, Hem1p, Hsp60p, Ilv6p, Lpd1p, Mkt1p, Nfs1p, Pbi2p, Pdc1p, Pgk1p, Pnc1p, Pro1p, Rax2p, Rib4p, Rpl23Ap, Rpl23Bp, Rps23Ap, Rps23Bp, Rps29Ap, Rps29Bp, Rps5p, Sec23p, Sec24p, Shm2p, Sod1p, Tpi1p, Tps3p, Vps1p, YDR348Cp, Yhb1p, YHL021Cp\eTD
          \eTR
            \bTR
            \bTD Mad2p--HPM\eTD
            \bTD\eTD
            \bTD 11/10\eTD
            \bTD\eTD
            \bTD 2\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Apl4p, Caf20p, Eno1p, Eno2p, Pdc1p, Pgk1p, Rrb1p, Trx2p, Ura7p, YOR283Wp\eTD
          \eTR
          \bTR
            \bTD Mcd1p--HPM\eTD
            \bTD\eTD
            \bTD 17/8\eTD
            \bTD\eTD
            \bTD 3\eTD
            \bTD\eTD
            \bTD Smc1p*,\crlf Trf4p\eTD
            \bTD\eTD
            \bTD Bdf1p, Csm1p, Nuf2p, Not5p, Pom152p, Srm1p, Stu2p, YBL005W--Ap, YDR170W--Ap, YMR045Cp, YNL284C--Bp, YNL284C--Ap, YMR046Cp\eTD
          \eTR
          \bTR
            \bTD Pds1p--HPM\eTD
            \bTD\eTD
            \bTD 4/1\eTD
            \bTD\eTD
            \bTD 1\eTD
            \bTD\eTD
            \bTD Esp1p*\eTD
            \bTD\eTD
            \bTD Azr1p, Ire1p, Mss1p, Swi3p\eTD
          \eTR
          \bTR
            \bTD Pds5p--HPM\eTD
            \bTD\eTD
            \bTD 0/1\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Mcd1p\eTD
            \bTD\eTD
            \bTD Aro4p, Chs5p, Hal5p, Kem1p, Mss1p, Pbp1p\eTD
          \eTR
          \bTR[bottomframe=on]
            \bTD Pho2p--HPM\eTD
            \bTD\eTD
            \bTD 4/1\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Rpl35Bp, Rpl35Ap, Rps5p, YBL005W--Ap, YDR170W--Ap, YDR261W--Bp, YGR161C--Cp, YJR026Wp, YOL103W--Ap, YML040Wp, YLR256W--Ap ,YLR227W--Ap, YLR157C--Ap, YJR028Wp, YMR045Cp, YNL284C--Bp\eTD
          \eTR
          \bTR[bottomframe=on,topframe=on]
            \bTD Pho4p--HPM\eTD
            \bTD\eTD
            \bTD 11/1\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Ade16p, Ade3p, Ape3p, Aro2p, Aro4p, Asn1p, Bbc1p, Bcy1p, Cct4p, Cct8p, Cdc33p, Cdc73p, Chs5p, Dbp2p, Dbp3p, Dig1p, Eap1p, Eft2p, Eft1p, Fas1p, Fun12p, Glk1p, Glt1p, Gly1p, Gua1p, Hef3p, Hom3p, Hrb1p, Hsp60p, Imd4p, Kem1p, Kri1p, Lys21p, Lys20p, Myo5p, Nfs1p, Nma1p, Nop1p, Nop58p, Nsr1p, Pab1p, Rpa135p, Rpa34p, Rpl11Bp, Rpl11Ap, Rpl12Bp, Rpl12Ap, Rpl16Ap, Rpl23Ap, Rpl23Bp, Rpl24Ap, Rpl24Bp, Rpl26Bp, Rpl26Ap, Rpl29p, Rpl34Ap, Rpl34Bp, Rpl35Bp, Rpl35Ap, Rpl36Ap, Rpl36Bp, Rpl38p, Rpl43Bp, Rpl43Ap, Rpl5p, Rpl7Ap, Rpl7Bp, Rpp1Ap, Rpp2Ap, Rpp2Bp, Rps12p, Rps19Bp, Rps19Ap, Rps2p, Rps23Ap, Rps23Bp, Rps25Ap, Rps25Bp, Rps27Bp, Rps27Ap, Rps29Ap, Rps29Bp, Rps5p, Rps7Ap, Rps7Bp, Rps9Ap, Rps9Bp, Rrb1p, Rrp5p, Rsp5p, Sec23p, Ses1p, Shm2p, Sik1p, Sin3p, Snf1p, Srm1p, Ste11p, Ste50p, Stm1p, Tsr1p, Tub1p, Tub2p, Tub3p, Ura7p, Utp7p, Vip1p, Vps1p, Vrp1p, YAR075Wp, YBL101W--Bp, YGR161W--Bp, YFL002W--Ap, YDR210W--Bp, YDR034C--Dp, YCL019Wp, YDR261W--Bp, YGL068Wp, YHR121Wp, YIL137Cp, YMR045Cp, YMR050Cp, YMR237Wp, YNL054W--Bp\eTD
          \eTR
          \bTR[topframe=on]
            \bTD Rtt102p--HPM\eTD
            \bTD\eTD
            \bTD 2/0\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Aro4p, Arp7p, Arp9p, Fyv6p, Gsy2p, Hsl1p, Hta2p, Hta1p, Htl1p, Ldb7p, Nfi1p, Nfs1p, Npl6p, Rim1p, Rpl35Bp, Rpl35Ap, Rpl36Ap, Rpl36Bp, Rpl43Bp, Rpl43Ap, Rps2p, Rps29Bp, Rrb1p, Rsc1p, Rsc2p, Rsc3p, Rsc4p, Rsc58p, Rsc6p, Rsc8p, Rsc9p, Sfh1p, Snf12p, Snf2p, Snf5p, Snf6p, Sth1p, Swi1p, Swi3p, Taf14p, YFL049Wp, YHR097Cp\eTD
          \eTR
          \bTR
            \bTD Sds22p--HPM\eTD
            \bTD\eTD
            \bTD 45/0\eTD
            \bTD\eTD
            \bTD 4\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Nip100p, Ppz1p, Snf1p, Stu1p, Vps8p, YBL010Cp\eTD
          \eTR
          \bTR
            \bTD Snf2p--HPM\eTD
            \bTD\eTD
            \bTD 164/13\eTD
            \bTD\eTD
            \bTD 11\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Chs5p, Pab1p, Rpl11Bp, Rpl11Ap, Rpl16Ap, Rpl26Ap, Rpl26Bp, Rpl34Ap, Rpl34Bp, Rpl35Bp, Rpl35Ap, Rpl36Ap, Rpl36Bp, Rps12p, Rps2p, Rtt102p, Sth1p, Stm1p, YDL053Cp, YGR161C--Cp\eTD
          \eTR
          \bTR
            \bTD Spo12p--HPM\eTD
            \bTD\eTD
            \bTD 18/5\eTD
            \bTD\eTD
            \bTD 1\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Act1p, Ado1p, Ahp1p, Ald6p, Azr1p, Bmh1p, Cpr1p, Cys3p, Eft2p, Eft1p, Eno1p, Eno2p, Gpm1p, Hsp12p, Hsp42p, Hxk2p, Pdc1p, Pgi1p, Pgk1p, Rhr2p, Rps12p, Rps19Bp, Rps19Ap, Tif2p, Tif1p, Trp3p, Trx2p, Yhb1p, YNL134Cp, YPL257W--Bp\eTD
          \eTR
          \bTR
            \bTD Yak1p--HPM\eTD
            \bTD\eTD
            \bTD 75/0\eTD
            \bTD\eTD
            \bTD 3\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Caf20p, Glt1p, Gly1p, Hef3p, Kem1p, Nfs1p, Rib4p, YJL206Cp\eTD
          \eTR
          \bTR[bottomframe=on]
            \bTD YHR115Cp--HPM\eTD
            \bTD\eTD
            \bTD 17/0\eTD
            \bTD\eTD
            \bTD 8\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Dbp3p, Gcd11p, Jip5p, Mkt1p, Sec16p, YBL101W--Bp, YLR410W--Bp, YGR161W--Bp, YFL002W--Ap, YDR210W--Bp, YDR034C--Dp, YCL019Wp, YJR026Wp, YOL103W--Ap, YML040Wp, YLR256W--Ap, YLR227W--Ap, YLR157C--Ap, YJR028Wp\eTD
          \eTR
          \bTR
            \bTD YNL116Wp--HPM\eTD
            \bTD\eTD
            \bTD 37/0\eTD
            \bTD\eTD
            \bTD 7\eTD
            \bTD\eTD
            \bTD ---\eTD
            \bTD\eTD
            \bTD Jip5p\eTD
          \eTR
        \eTABLEbody
      \eTABLE
    }
    Of the original set of 25 gene products that we set out to tag and purify, 21 yielded utilizable results. We were unable to amplify the HPM--cassette with primers to tag CDC5 and ESS1 while TAP--MudPIT experiments for Bir1p--HPM and Nbp1p--HPM resulted in little or no recovery of the tagged baits themselves. Of the 21 <qu>successful</qu> purifications that yielded sequence coverage for the tagged bait, 20 of the experiments (95\%) yielded interacting proteins that are either true binding partners validated by other direct approaches, probable binding partners that display genetic interaction with the bait, or candidate binding partners that were found in association with only one bait. The Pho2p--HPM experiment yielded `hits' only from proteins that were found associated with other, unrelated baits or were otherwise deemed to be likely contaminants.\par
    The set of bait proteins evaluated in this study overlaps considerably with the Ho et al. ((2002) effort. Figure x.x compares the retrieval of physical interactors for 13 gene products used as baits in both studies. Notably, in each case our approach identified at least as many or more of the previously--known binding partners of the 13 bait proteins. For eight of the baits, Ho et al. (2002) identified more putative interacting partners. However, since Ho et al. (2002) utilized single--step affinity purification of overproduced bait protein, additional interactions revealed only in that study should be considered as tentative, pending verification by independent methods.\par
    The second issue that we addressed was the feasibility of using a filtering approach to cull nonspecific contaminants from the list of proteins identified in each TAP--MudPIT experiment. The idea is that nonspecific proteins should show up in a high fraction of experiments, whereas specific interactors should only show up in one or a small number of experiments (depending upon the degree of functional relatedness of the tagged genes in the query set). We found that proteins that were identified in five or more TAP--MudPIT experiments tended to have a high codon adaptation index (Sharp et al. 1987), which is a rough measure of abundance (Jansen et al. 2003, data not shown). Based on this correlation, we automatically considered proteins found in more than five experiments to be probable contaminants. A similar filtering approach was employed by Gavin et al. (2002) and Ho et al. (2002), but since their data--sets were much larger they were able to employ lower thresholds.\par
\stoptext