Efﬁcient Elution from Antibody-Conjugated Afﬁnity Beads research articles Figure 3. UV chromatograms of proteolyzed cross-linked and noncross-linked antibodies analyzed using a monolithic column. Cohesin was puriﬁed using RRAD21 antibody beads. (a) Cross-linked and noncross-linked RRAD21 antibody beads were incubated with LysC for 15 min and an aliquot, which corresponded to 2 pmol antibodies (assuming complete cleavage) was separated using a monolithic column. (b) 50-fold the amount shown in (a) (of cross-linked RRAD21 antibody beads) was separated using a monolithic column, after 15, 30, and 60 min of incubation with LysC. For better illustration chromatograms are displayed with a 5% time and a 15% signal offset. lanes on the protein gel with respect to the elution efﬁciency. However, the 2- to 3-fold more intense XICs after LysC elution correspond well with the tightly bound fraction not eluted using the acidic buffers, suggesting greatly improved if not complete elution after only 1 h of LysC predigestion. Proteolytic stability tests described above using a monolithic column revealed that no or only a minor amount of antibody is cleaved during the 1 h LysC incubation (Figure 1). However, to verify this result and to compare the degree of sample contamination from the antibody in acid elution versus LysC elution we extracted XICs of identiﬁed peptides stemming from the Fc region of the IgG (3 identiﬁed in glycine eluate, 7 in LysC eluate of APC/C puriﬁcation using RAPC3 antibody). XICs of the LysC eluate were on average 20-fold more intense compared to glycine eluate (3 peptides in common) (Figure S5, Supporting Information). Similar results were obtained when the contamination from RRAD21 antibody peptides was evaluated (not shown). But in general, antibody peptides accounted for only a minor amount of the TIC (total ion current) in the glycine and protease eluted sample, respectively (Figure S5, Supporting Information). Hence the protease elution protocol allowed the complete recovery of immunoadsorbed protein complexes after only 1 h of incubation with LysC with only very little contamination from antibody peptides. Can Noncross-Linked Antibody-Conjugated Beads Be Used? Chemical cross-linking not only covalently attaches the antibody to the Protein A Afﬁprep beads but also introduces intramolecular cross-links within the antibody. This can affect the binding sites leading to a reduction or even loss in speciﬁcity toward the antigen. Therefore, we tested whether noncross-linked antibody-conjugated beads are also compat- ible with the protease elution method. We puriﬁed cohesin using cross-linked and noncross-linked antibody beads and performed LysC elution. To monitor whether the antibody is intrinsically stable to proteolysis or whether chemical cross- linking confers resistance we separated LysC predigested aliquots of the 15 min, 30 and 60 min time-point using a monolithic column. As performed in the initial proteolytic stability experiments we again loaded a volume that cor- responded to 2 pmol of antibody (assuming complete diges- tion). Figure 3a shows UV absorption of the 15 min time point in the range of 1.5-3.5 mAU in case of the noncross-linked antibody and lesser then 0.3 mAU in case of the cross-linked antibody. As 1 pmol digested BSA separated on the same column gives absorption in the range of 2.5-5 mAU (Figure S1, Supporting Information) it can be concluded that the noncross-linked antibody is rapidly predigested and therefore contaminates the sample to an extent unsuitable for further MS analysis. In contrast, when chemically cross-linked, the antibody was again shown to be resistant to proteolysis. To monitor the kinetics of sample protein release from cross-linked antibody-beads we separated a 50-fold greater amount of the sample from the 15 min, 30 and 60 min predigestion time- point again using a monolithic column. Figure 3b shows only a minor increase in UV absorption from the 15 min to the 60 min time-point, indicating rapid predigestion and release of proteins. Subsequent LC-MS/MS analyses revealed that the number of unique peptides identiﬁed (Mascot score > 25) from the core cohesin subunits was essentially identical in all three predigestion periods analyzed (Table S4, Supporting Informa- tion). Hence, mass spectrometry based identiﬁcation conﬁrmed the interpretation of the results obtained with the monolithic column. Proteins bound to the afﬁnity beads are sufﬁciently predigested already after 15 min leading to the release of proteins from the afﬁnity beads. Absolute Quantiﬁcation of Cohesin Subunits and Stoichiometry Calculation. To test the robustness and com- patibility of the protease elution protocol with AP-QMS strate- gies we performed absolute quantiﬁcation of core cohesin subunits, tested whether different protease predigestion periods inﬂuence the outcome of stoichiometry determination and compared the obtained results to a standard acid elution protocol. Journal of Proteome Research Vol. 10, No. 2, 2011 785