Efﬁcient Elution from Antibody-Conjugated Afﬁnity Beads ner(s). It makes use of a speciﬁc antibody-antigen interaction and therefore does not necessitate any artiﬁcial sequence (tag) incorporated into the “bait” which could interfere with the protein’s function such as the assembly into a macromolecular protein complex. Traditionally proteins are eluted from the antibody beads using glycine at low pH. Chemical labeling with reagents that target primary amines such as iTRAQ, TMT or mTRAQ for relative and absolute quantiﬁcation are incompat- ible with the use of glycine.17-19 Therefore protein elution is performed with diluted acids such as HCl, TFA or FA at concentrations of ∼100 mM if chemical labeling using these reagents is to be performed. Independent of the acid elution protocol used incomplete elution from the antibody-conjugated beads is frequently observed. As a direct consequence, less material is available for the LC-MS analysis, leading to lower sequence coverage and lower signal-to-noise ratio in quanti- ﬁcation experiments, respectively. Labugger et al. and Chres- tensen et al. established an “on-bead digestion” protocol, which led to an increase in sequence coverage in the study of their protein of interest.20,21 In this protocol proteins are reduced, alkylated and subsequently digested for a prolonged period of time (usually overnight) while still bound to the antibody- conjugated beads. Although this protocol has been used in a number of studies7,20-25 several important parameters/factors have not been tested yet; (i) the sample contamination from cleaved antibody peptides when using cross-linked and non- cross-linked antibody-conjugated beads, respectively, (ii) a comparison of acid elution methods to the on-bead digestion in a quantitative fashion, (iii) the time period required for efﬁcient elution by the protease. Furthermore, in light of the recent shift toward quantitative interaction studies, there is a need to test the compatibility of the on-bead digestion protocol with quantiﬁcation strategies such as the absolute quantiﬁca- tion of protein complex subunits and the subsequent calcula- tion of complex stoichiometry. As a model complex for stoichiometry determination based on absolute quantiﬁcation, we selected the cohesin complex. Cohesin’s main function is to hold sister chromatids together from their synthesis in S-phase of the cell cycle until the metaphase to anaphase transition, where cohesin is removed to allow separation of sister chromatids and exit from mitosis.26,27 Cohesin is an evolutionary well conserved protein complex consisting of four core components; SMC1, SMC3 and RAD21 form a ring-like structure to which STAG binds (reviewed in ref 28). The exact subunit stoichiometry is not known, but on the basis of electron micrographs and biochemical experiments, a 1:1:1:1 stoichiometry has been proposed. Adding to the cohesin complexity, in somatic cells STAG occurs in two isoforms (STAG1, STAG2) which associate with the cohesin subunits in a mutually exclusive manner.29,30 In other words, cohesin complexes exist in two different populations; STAG1 and STAG2 containing cohesins, respectively. In human cul- tured HeLa cells, STAG2 was shown to be more abundant than STAG1, but the exact ratio of STAG1:STAG2 containing cohesins is not known.29,30 Here we show by labelfree and absolute quantiﬁcation that a short 15-60 min predigestion with LysC (modiﬁed on-bead digestion termed protease elution) is 2- to 3-fold more efﬁcient than the acid elution protocols. Using 19 reference peptides generated with our EtEP strategy,31 of which 6 are speciﬁc for STAG1 and STAG2, respectively, we determined the cohesin complex stoichiometry and the exact STAG1/STAG2 stoichi- ometry from exponentially growing HeLa cells. research articles Methods and Materials General Chemicals and Reagents. All chemicals purchased were of highest purity available. Triﬂuoroacetic acid (TFA), hydrochloric acid (HCl), isopropanol and tris(2-carboxyeth- yl)phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich (Steinheim, Germany). Formic acid (FA) was obtained from SAFC Biosciences (Andower, U.K.); triethlyam- monium bicarbonate (TEAB) and S-methyl thiomethane- sulfonate (MMTS) from Fluka (Buchs, Switzerland); and MS- grade modiﬁed trypsin was purchased from Promega (Madison, WI). Ultrapure 18-MΩ water was obtained from a Millipore Milli-Q-system (Bedford, MA). Generation of mTRAQ Heavy Labeled Internal Reference Peptides. Generation of reference peptides was performed essentially as described.31 In brief, peptides were synthesized by solid-phase Fmoc chemistry and puriﬁed on a Vision HPLC instrument (Applied Biosystems, Foster City, CA). According to the EtEP strategy, peptides were then trypsinized, labeled with mTRAQ heavy (ABSCIEX, Foster City, CA), quantiﬁed via mTRAQ light labeled equalizer peptide (GVTASVAGAR, amino acid analyzed) and mixed in equimolar amounts (Figure S6, Supporting Information). Binding of Antibodies to Beads. For immunoadsorption experiments, afﬁnity-puriﬁed antibodies were coupled to Af- ﬁprep protein A beads (Bio-Rad, Hercules, CA) in a ratio of 1 mg antibodies to 1 mL beads. For puriﬁcation of cohesin we used an antibody raised against peptide FHDFDQPLPDLD- DIDVAQQFSLNQSRVEEC of RAD21 (antibody 575) and for puriﬁcation of ACP/C we used an antibody raised against peptide CTDADDTQLHAAESDEF of APC3 (antibody 233). Cross-linking 300 µ L beads with 300 µ g antibody was performed in 20 mM DMP (dimethyl pimelimidate dihydrochloride; Sigma-Aldrich, Steinheim, Germany) in 0.2 M Na-borate pH 9.2 for 30 min at RT. After two wash steps with 3 mL (10 bead volumes (bv)) 0.2 M Tris-HCl at pH 8.0 antibody-conjugated beads were washed twice with 3 mL (10 bv) of 0.1 M glycine at pH 2.0 to remove noncross-linked antibodies and ﬁnally washed with 3 mL (10 bv) of 1× PBS. Immunoadsorption of Cohesin and APC/C. HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum, 0.2 mM L-glutamine and antibiotics (all Invitrogen, Carlsbad, CA). For each immunoadsorption cells from four trays (25 × 25 cm2, ∼70% conﬂuent) were harvested, washed once in 1× PBS and lysed with 15 strokes using a dounce homogenizer in 6 mL lysis buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 10% glycerol, 1% triton X100, 2 mM EDTA and protease inhibitor cocktail mix). For binding, HeLa total cell extract S-20 was incubated with 30 µ L antibody-conjugated beads and incubated on a rotary shaker for 90 min at 4 °C. Subsequently, the beads were washed 6 times with 1.5 mL (50 bv) of wash buffer (as lysis buffer but 1% trition X-100 was substituted for 0.2% NP- 40). Finally, before elution beads were washed with 1.5 mL (50 bv) of 150 mM NaCl, pelleted and the supernatant was carefully removed. Acid Elution, Protease Elution and Complex Digestion. For acid elution, 15 µ L beads were incubated on a rotary shaker for 3 min at RT with 30 µ L (2 bv) of 100 mM glycine pH 2.0, 100 mM HCl, 100 mM FA and 100 mM TFA, respectively. The supernatant was collected and neutralized by adding 1.5 µ L (0.1 bv) 2 M NaOH. Finally, the eluate was diluted 1:2 with 1 M TEAB. For protease elution, 15 µ L beads were resuspended with 30 µ L (2 bv) of 500 mM TEAB and incubated with 500 ng Journal of Proteome Research Vol. 10, No. 2, 2011 781