Efﬁcient Elution from Antibody-Conjugated Afﬁnity Beads Figure 5. Relative abundance of STAG isoforms in the population of cohesin complexes. Cohesin is predicted to form a ring-like structure (ref 28) with a subunit stoichiometry of 1:1:1:1 as measured in this study. The relative abundance of STAG1 containing cohesin is 6-8% and that of STAG2 is 92-94% in soluble extract of logarithmically growing HeLa cells. method. Calculation of peptide amount was performed by averaging the light/heavy ratios of all three transitions moni- tored. Due to the low abundance of STAG1, upon HCl elution all three transitions of the peptide YSADAEK were below the threshold, not allowing quantiﬁcation (not shown). Intensity of EDLLVLR speciﬁc transition 499.3f710.4 (b5 fragment) was below the 500 cps threshold, leaving only two transitions for quantiﬁcation (Figure S10, Supporting Information). Addition- ally, the calculated absolute amount of STAG1 loaded onto the column was 330 amol and was thus below the tested dynamic range of STAG1 speciﬁc reference peptides. In contrast, due to the 3-fold better recovery of the protease elution all YSADAEK and EDLLVLR transitions were above the 500 cps threshold allowing quantiﬁcation of STAG1 based on all 3 spiked reference peptides. The measured absolute amount of STAG1 was 820 amol and therefore within the tested dynamic range. In summary, the experiments illustrate the superiority of the protease elution method over standard acid elution with respect to recovery from afﬁnity beads. Furthermore, they demonstrate the robustness and also the compatibility of the protease elution protocol with AP-QMS strategies such as the stoichiometry determination of protein complexes. Stoichiometry determination based on the absolute quan- tiﬁcation of interacting subunits requires puriﬁcation of a homogeneous sample.12 We selected a RRAD21 antibody for coimmunoadsorption experiments because most if not all RAD21 is found in the 14S cohesin complex, where other subunits have also been detected in subcomplexes of cohesin. For example, SMC1 and SMC3 also exist as a heterodimer.29 Thus, using a RAD21 antibody allowed us to separate both of the 14S cohesin complexes (STAG1 and STAG2 containing) from cohesin subcomplexes without enriching for free, non cohesin associated RAD21. In the present study we showed for the ﬁrst time that cohesin from soluble HeLa extract forms a complex with a 1:1:1:1 stoichiometry. This is in agreement with the current structural model of cohesin based on electron micrographs, crystal structure of subunit fragments and avail- able biochemical data (reviewed in ref 28). Only a few studies so far reported the quantiﬁcation of protein isoforms.35-38 We used a total of 7 reference peptides for STAG quantiﬁcation; three were speciﬁc for isoform 1, three were speciﬁc for isoform 2 and one measured both isoforms (DGIEFAFK). In all cases measured amounts of DGIEFAFK were within a 10% tolerance of STAG1 + STAG2 amounts, thus research articles increasing the conﬁdence of STAG quantiﬁcation (Figure S8). It was previously shown by immunoadsorption and Western blot analysis that the ring-like SMC1-SMC3-RAD21 structure preferentially associates with STAG2. The authors concluded from their experiments that the STAG1:STAG2 ratio of cohesin subpopulations is about 1:3 (see Figure 3D in ref 30). Here, using MS based absolute quantiﬁcation we determined the STAG1:STAG2 ratio to be in the range of 1:12 to 1:15 in cohesin puriﬁed from soluble extract of exponentially growing HeLa cells (Figure 5). The discrepancy could be explained by the limited dynamic range and the semiquantitative nature of quantiﬁcation based on Western blotting and densitometry.39 Conclusion In the present study, we have shown that a protease mediated elution increased the recovery of cohesin and APC/C from antibody-conjugated afﬁnity beads by a factor of 2 to three. Hence, while acid elution was found to be less efﬁcient, a simple predigestion for 15-60 min was sufﬁcient for greatly improved if not complete recovery without leading to a signiﬁcant sample contamination from cleaved antibody pep- tides. Furthermore, absolute quantiﬁcation and subsequent stoichiometry determination of the cohesin complex demon- strated the robustness and compatibility of the protease elution method with AP-QMS studies. Using this elution technique we have for the ﬁrst time measured the subunit stoichiometry of the cohesin complex from soluble extract of HeLa cells. The protease elution method is ideally suited for absolute quanti- ﬁcation studies where sensitivity is crucial, demonstrated in this study by measuring the exact STAG1:STAG2 stoichiometry within the population of cohesin complexes. It can further be applied to the determination of protein copy numbers due to efﬁcient if not complete recovery from afﬁnity beads (if complete depletion of the protein from the extract is shown). Applied to the identiﬁcation of protein complexes in AP-QMS studies it has the potential to increase the sensitivity for detecting low abundant interactors, again due to higher recovery compared to acid elution and because of no or only insigniﬁcant contamination from cleaved antibody peptides compared to standard on-bead digestion. Acknowledgment. We are grateful to our colleagues from the Mechtler and the Peters Group: Elisabeth Roitinger, Andreas Schmidt and Thomas Ko¨cher for useful discussions and critical reading of the manuscript, Jim Hutchins for help with immunoadsorption, Venugopal Bhaskara for generously providing antibodies and for useful discussions. Work in the Mechtler and Peters Group is supported by Boehringer Ingelheim, the Austrian Proteomics Platform (APP-III) within the Austrian Genome Research Programme (GEN-AU III) and by the MitoSys project funded by the 7th framework program of the European Union. J.H. was supported by the Austrian Science Foundation (FWF), SFB-F3402, Chromosome Dynamics. Supporting Information Available: Additional Results and Discussion, monolithic chromatograms, details on the quantiﬁcation by SRM are given. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sullivan, M.; Morgan, D. O. Finishing mitosis, one step at a time. Nat. Rev. Mol. Cell Biol. 2007, 8 (11), 894–903. Journal of Proteome Research Vol. 10, No. 2, 2011 787