research articles LysC (Wako, Richmond, VA) or trypsin (MS grade trypsin gold, Promega), respectively for the time periods indicated in the results and discussion section. After elution by predigestion the supernatant was collected and diluted with 500 mM TEAB to give the same ﬁnal volume as after acid elution. Next, differently eluted fractions were reduced in 1 mM TCEP for 30 min at 56 °C and alkylated in 2 mM MMTS for 30 min at RT in the dark. Samples were then further digested by addition of 500 ng of LysC for 14 h at 37 °C followed by a 16 h digest with 1 µ g trypsin at 37 °C (in case of LysC elution) or digested by addition of 500 ng trypsin and incubation for 16 h at 37 °C (in case of trypsin elution). Digested samples were then either analyzed on an Orbitrap XL for labelfree quantiﬁcation or labeled with mTRAQ heavy (HCl elution) according to the manufacturer’s instructions for absolute quantiﬁcation on a 5500 QTRAP instrument. To assess completeness of mTRAQ labeling a fraction of the labeled sample was analyzed on an Orbitrap XL and spectra were searched against the IPI database using Mascot 2.2 with and without mTRAQ light as variable modiﬁcation. Analysis on nano-HPLC Equipped with a Monolithic Column. Thirty µ L RAPC3 and RRAD21 antibody-conjugated beads were suspended with 60 µ L (2 bv) 500 mM TEAB and incubated with 500 ng trypsin and 500 ng lysC, respectively. After incubation for 1, 4, and 16 h at 37 °C under constant shaking at 1300 rpm in an Eppendorf Thermomixer, 10 µ L of the supernatant were collected and proteolysis was stopped by addition of 240 µ L 1% TFA. Then 1.5 µ L sample diluted in 20 µ L 0.1% TFA was separated on a Dionex Ultimate equipped with a 200 µ m I.D. monolithic column (PepSwift from Dionex, Amsterdam, The Netherlands) using a gradient from 5% ACN, 0.1% TFA to 80% ACN, 10% TFE, 0.08% TFA over 25 min. Labelfree Quantiﬁcation on Orbitrap XL. One ﬁfth of each eluate was separated using a Dionex Ultimate 3000 nanoLC system equipped with a C18 PepMap column (75 µ m i.d. × 150 cm length, 3 µ m particle size, 100 A pore size) (Dionex, Amsterdam, The Netherlands) using a 55 min gradient from 5% ACN, 0.1% FA to 30% ACN, 0.1% FA followed by a 5 min gradient from 30% ACN, 0.08% FA to 80% ACN, 10% TFA, 0.08% FA. Peptides eluting from the nano-RP-HPLC were analyzed on an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientiﬁc, Bremen, Germany), equipped with a nanoelectro- spray ion source (Proxeon, Odense, Denmark) with an applied voltage of 2 kV. The mass spectrometer was operated in data- dependent mode: 1 full scan (m/z 350-1600) was acquired in the Orbitrap (resolution of 60 000) followed by MS/MS scans of the ﬁve most abundant ions in the LTQ. The chosen ions were excluded from further selection for 60 s. Fragment ion data were interpreted using Mascot 2.2 (Matrix Science, London, UK) within the Proteome Discoverer Software (Thermo Fisher Scientiﬁc, v 1.2). Data were searched against the human International Protein Index (IPI) database (v 3.74). Following search parameters were used: tryptic peptides; up to 3 missed cleavage sites; oxidation (M), phosporylation (S,T,Y), pyro- glutamate (N-term) as variable modiﬁcations and methylthio (C) as ﬁxed modiﬁcation; peptide mass tolerance of 3 ppm and fragment ion tolerance of 0.5 Da. Extracted ion chromatograms (XICs) of peptides with a Mascot score of g25 and which were ranked 1 were extracted using the Precursor Ion Area Detector feature within Proteome Discoverer 1.2 with a mass tolerance of 3 ppm. For labelfree quantiﬁcation ratios of XICs of peptides identiﬁed with all 5 elution methods were calculated relative to glycine elution. Only peptides without chemical modiﬁcation 782 Journal of Proteome Research Vol. 10, No. 2, 2011 Holzmann et al. (such as oxidized methionine, S,T,Y-phosphorylation, N-ter- minal pyroglutamate) were included in relative quantiﬁcation. Absolute Quantiﬁcation by SRM Analysis on a 5500 QTRAP. Immediately before LC-SRM analysis digested and labeled cohesin was spiked with 25 fmol of each internal reference peptide in case of the experiment shown in Figure 4a and with 10 fmol in case of the experiment shown in Figure 4b. To remove excess of 2-propanol samples were concentrated in a Speed Vac for 10 min to a ﬁnal volume of approximately 25% of the starting volume and rediluted with 0.1% TFA to identical sample volumes. Samples were then separated on a Dionex Ultimate nano-HPLC equipped with a C18 PepMap column (75 µ m ID × 150 mm length, 3 µ m particle size, 100 Å pore size) (Dionex, Amsterdam, The Netherlands) using the following gradient of solvents A (5% ACN, 0.1% FA), B (30% ACN, 0.08% FA) and C (80% ACN, 10% TFE, 0.08% FA) at a ﬂow rate of 300 nL/min: from 0% B, 0% C to 100% B, 0% C over 30 min followed by a gradient to 0% B, 90% C over 5 min. Peptides eluting from the nanoLC were analyzed on a 5500 QTRAP instrument (ABSCIEX, Foster City, CA) equipped with a nanoelectrospray source with applied voltage of 2.3 kV. The mass spectrometer was operated in scheduled SRM mode with the following parameters: MRM detection window of 180 s, target scan time of 2 s, curtain gas of 20, ion source gas 1 of 15, declustering potential of 75, entrance potential of 10. Q1 resolution was set to unit and Q3 resolution to low. Pause between mass ranges was set to 2.5 ms. Three SRM transitions per peptide (Table S1, Supporting Information) were selected and optimized for collision energy by direct infusion of internal reference peptides. Collision cell exit potentials (CXP) were calculated by dividing Q3 mass by a factor of 29. Peak integration was performed using MultiQuant 1.2 (ABSCIEX, FosterCity, CA) software and manually reviewed. Light to heavy peak area ratios were calculated over the three transitions and two replicates to calculate absolute amounts loaded on column and complex stoichiometry. Results and Discussion Testing the Proteolytic Stability of Cross-Linked Antibody- Conjugated Beads. In the standard on-bead digestion protocol antibody-conjugated beads are incubated for a prolonged period of time with a protease to allow digestion of the puriﬁed sample.20-25,32,33 To test the proteolytic stability of the cross- linked antibody beads and therefore to assess a potential sample contamination from antibody peptides, we incubated Afﬁprep protein A sepharose beads cross-linked with two different antibodies (RAPC3 and RRAD21) with trypsin and LysC, respectively. After 1, 4, and 16 h of incubation, beads were removed by centrifugation and an aliquot of the super- natant was separated using a monolithic column, which allows the simultaneous separation of peptides and proteins (Figure 1). We loaded a volume of the supernatant that corresponded to 2 pmol of antibody (assuming complete proteolysis and hence release of antibody peptides to the supernatant). After 1 h of incubation with trypsin and LysC, respectively no signals other than background were visible in the UV chromatograms, indicating no or only little cleavage of the antibodies. In case of trypsin, after 4 h of incubation the ﬁrst additional signals were visible, indicating minor cleavage and after 16 h signal intensities stemming from cleaved antibodies were further increased (compare to 1 pmol of trypsinized BSA shown in Figure S1, Supporting Information, and to Figure 3a). In contrast to incubation with trypsin, antibody beads incubated