Efﬁcient Elution from Antibody-Conjugated Afﬁnity Beads research articles Figure 1. UV chromatograms of proteolyzed cross-linked antibodies analyzed using a monolithic column. RRAD21 (panels (a) and (b)) and RAPC3 (panels (c) and (d)) antibodies cross-linked to AfﬁPrep A beads were incubated with trypsin and LysC, respectively. At the indicated time points an aliquot, which corresponded to 2 pmol antibody (assuming complete cleavage) was separated using a monolithic column. Increasing absorption in the UV chromatograms indicate proteolytic cleavage of the antibodies. For better illustration, chromatograms are displayed with a 5% time and a 15% signal offset. with LysC resulted in no, or only minor, cleavage (after 16 h, RRAD21) of the antibody. The observed higher resistance of the antibodies toward proteolysis when incubated with LysC can most likely be explained by two factors: (i) the trypsin we used is chemically modiﬁed to inhibit autolysis and to increase stability whereas the unmodiﬁed LysC undergoes autolysis and therefore loses activity during incubation and (ii) trypsin cleaves C-terminal to arginine and lysine whereas LysC only cleaves after lysine, which in turn is chemically modiﬁed by the cross- linker. Hence, cross-linking results in one recognizable cleavage site for trypsin but no site for LysC on the surface of the antibody. In none of the studies that employed on-bead digestion a potential contamination from cleaved antibody peptides was assessed, although proteolysis of cross-linked antibodies was described.34 Due to the fact that the antibody is usually present in excess compared to the puriﬁed sample, already minor antibody cleavage, as observed after 4 h of proteolysis, can lead to a signiﬁcant increase of background unfavorable for subse- quent LC-SRM or LC-MS/MS analyses. To test the inﬂuence of antibody proteolysis on MS-based identiﬁcation we puriﬁed cohesin and APC/C and performed on-bead digestion for 1, 4 and 16 h followed by a sequential digestion after separation of beads and sample. MS analysis on an LTQ Orbitrap revealed that the number of peptides identiﬁed (Mascot score >25) was highest after only 1 h of on- bead predigestion for elution compared to 4 and 16 h, regard- less of the protease used for on-bead predigestion (Table S2, Supporting Information). For example, the known cohesin interactor Wings apart-like (WAPL) was identiﬁed with 11 peptides after 1 h LysC elution, with 8 peptides after 4 h LysC elution and with only 3 peptides after 16 h LysC elution. In case trypsin was used for elution this effect was even more pronounced; we identiﬁed 9 WAPL peptides after 1 h, 1 peptide after 4 h and no peptide was identiﬁed after 16 h predigestion for elution. MS analysis of APC/C eluted with 1, 4 and 16 h on-bead predigestion showed a similar result (Table S3, Sup- porting Information). These effect most likely results from two factors: (i) the mass spectrometer spends time on sequencing antibody peptides and more importantly (ii) extended on-bead digestion periods result in a dramatically increased protein concentration due to released antibody peptides (Figure 1), which limit the amount of sample that can be loaded onto the column (see Tables S3 and S4 for further details, Supporting Information). For example, of the 16 h trypsin on-bead predigested cohesin sample we could only load 1/5th of the volume compared to the 1 h on-bead predigestion, nevertheless the total amount of protein loaded was considerable higher and close to the columns capacity as judged by the UV chromatogram (not shown). Consequently, to limit the degree of antibody proteolysis and to therefore increase sensitivity, we decided to shorten codigestion of sample and antibodies to 1 h followed by a sequential digestion of the sample after Journal of Proteome Research Vol. 10, No. 2, 2011 783