research articles Holzmann et al. Figure 2. (a) SDS-PAGE of isolated APC/C and cohesin from soluble extract of HeLa cells. (b) Labelfree quantiﬁcation of cohesin subunits eluted using different protocols by XIC relative to glycine elution. (c) Labelfree quantiﬁcation of APC/C subunits eluted using different protocols by XIC relative to glycine elution. Data are mean ( SD of duplicate measurements. See also Figures S3 and S4 for peptide scattering within subunits in the technical duplicates (Supporting Information). separation from antibody-conjugated beads. Hence we refer to this protocol as “protease elution” rather than “on-bead” digestion. Afﬁnity Puriﬁcation of Cohesin and APC/C and Comparison of Elution Protocols by Labelfree Quantiﬁcation. To test such a “protease elution” protocol in terms of elution efﬁciency and sample contamination from cleaved antibody peptides we puriﬁed cohesin and APC/C by immunoadsorption from soluble extract of HeLa cells, performed labelfree quantiﬁcation on an Orbitrap XL and compared the results to standard acid elution protocols. Protein complexes were puriﬁed using Af- ﬁprep beads cross-linked with RRAD21 and with RAPC3 antibodies, respectively and eluted using glycine at pH 2.0 (Figure 2a). Analysis on an Orbitrap XL and Mascot searches of the acquired spectra against the IPI database (v 3.74) identiﬁed the four core cohesin subunits within the top 8 identiﬁcations and 12 out of 13 APC/C subunits within the top 50 identiﬁcations, respectively. In order to allow a quantitative comparison of different elution methods we puriﬁed cohesin and APC/C and performed XIC (extracted ion chromatogram) based labelfree quantiﬁcation on an LTQ Orbitrap XL instru- ment. Immediately before the elution step, Afﬁprep beads were split into 5 aliquots and proteins were eluted using 2 bead volumes (bv) of the following reagents; 100 mM glycine pH 2.0, 100 mM HCl, 100 mM FA and 100 mM TFA (Figure S2, Supporting Information). Additionally we resuspended 1 aliquot of beads in 2 bv of 500 mM TEAB buffer and added LysC protease. We selected LysC protease for elution because our standard digest protocol for quantiﬁcation studies is comprised of a sequential LysC-trypsin double digestion. Additional results obtained using trypsin for elution is presented in the Supporting Information. In the on-bead digestion protocol 784 Journal of Proteome Research Vol. 10, No. 2, 2011 proteins bound to the antibody beads are incubated with a proteaseforaprolongedperiodoftime,usuallyovernight.20-25,32,33 In contrast to standard on-bead digestion and based on the proteolytic stability experiments discussed above we incubated the antibody beads for only 1 h at 37 °C with LysC, arguing that a short predigestion of the bait protein might be sufﬁcient to release the protein complexes from the antibody beads. We therefore refer to this protocol as “protease elution” rather than “on-bead digestion”. Supernatants were collected and subse- quently digested as described in the materials and methods section using a LysC-trypsin double digest and identical sample volumes were analyzed on an Orbitrap XL instrument. Spectra were searched against the human IPI database using the Mascot search engine and ion chromatograms (XIC) were extracted through Proteome Discoverer 1.2 software. Labelfree quantiﬁcation based on XICs of cohesin peptides identiﬁed in all 5 samples revealed that elution using HCl, FA and TFA was less efﬁcient than standard glycine elution, while XICs of LysC eluted peptides were on average 3-fold higher compared to glycine elution (Figure 2b and Figure S3, Supporting Informa- tion). Labelfree quantiﬁcation of APC/C subunits showed a similar result; HCl, FA and TFA elution were equally or slightly less efﬁcient, while LysC elution was on average 2-fold more efﬁcient compared to standard glycine elution (Figure 2c and Figure S4, Supporting Information). SDS-PAGE of eluate frac- tions and the supernatant after boiling the beads in SDS containing buffer (Laemmli buffer) revealed that a considerable portion of the puriﬁed protein complexes were not eluted by the acidic buffers (Figure S2, lanes denoted as “beads”, Sup- porting Information). Using the protease elution protocol, protein complexes are already predigested to smaller fragments. Therefore no conclusion can be drawn from the corresponding