The 85% value is not statistically different from the 91 17% for saturation PSA binding for IgG2a attached to Au nanoparticles by Protein A, estimated by an enzymatic method.9 This control suggests that the antibodies bound on MBs via protein A are intact and not significantly denatured. DISCUSSION The results above show that restricted proteolysis using MB-trypsin coupled with LC-MS/MS proteomic analysis permits direct evaluation of the orientation of surface-immobilized antibodies. from VL domain name, Fab region; Pep 5 (TSTSPIVK) from CL domain name, Fab region; Pep 8 (DLPAPIER) from CH2 domain name, Fc region; and Pep 11 (TELNYK) from CH3 domain name, Fc region. These four representative peptides all gave excellent linear calibration plots of peak area ratio of each representative peptide to internal standard vs peptide concentration (Physique 3). The different slopes are due to the different electrospray ionization efficiencies and variable fragmentation patterns of each peptide.33 Open in a separate window Determine 3. Calibration plots of normalized peak areas ratio for four representative peptides from mouse IgG2a vs peptide concentration. Error bars are standard deviations for = 3. Trypsin and MB-Trypsin Digestion on Immobilized Antibodies. Antibodies (Ab) were specifically captured by MB-ProA/G conjugates via Fc regions to give oriented antibodies (MB-ProA/G-Ab) or covalently conjugated to carboxylated MBs via EDC/NHSS-based amine-to-carboxyl nonoriented conjugation (MB-Ab, Scheme 1). Amounts of antibodies on MB-Ab and MB-ProA/G Epithalon conjugates were estimated using Micro BCA Protein Assay (Physique S4A). The molecular weight of mouse IgG antibody is usually ~150 kDa, and 20 mg MB corresponds to 2.0 109 particles (according to vendor); 64 000 6000 antibody molecules were estimated on each MB-ProA/G-Ab, while 59 000 7000 antibody molecules were estimated on each MB-Ab, which is in good accord with a previous report.25 No statistically significant difference of antibody numbers per bead was found between MB-ProA/G-Ab and MB-Ab using a > 0.05. We first used free trypsin in answer for proteolysis of MB-ProA/G-Ab and MB-Ab conjugates under nondenatured conditions, and the resulting peptide mixture was analyzed by LC-MS/MS. Plateaus in the MRM LC peak intensity were found between 6 and 12 h of reaction (Physique 4), indicating that digestion is usually complete. Thus, the peptide Epithalon concentrations at each Epithalon time interval were calculated using calibration plots (Physique 3) and compared with those at 12 h (defined as 100%). For the MB-ProA/G-Ab conjugates, a significantly larger digestion rate in the initial 3 h stage was found for Pep 2 (YASQSISGIPSR) from VL domain name, Fab region (Figures 4A and S5A), indicating the variable domains of the immobilized IgG2a antibody were oriented away from the MB and toward the trypsin answer due to Ab-stem linkage to the MB surface around the Fc-binding protein A/G. For MB-Ab conjugates, no significant difference among the digestion rates of the four representative peptides was found (Physique 4B), consistent with randomly immobilized IgG2a comparable opportunities for trypsin digestion of Fc and Fab domains. Open in a separate window Physique 4. Free trypsin digestion kinetic results over 12 h for (A) MB-ProA/G-Ab and (B) MB-Ab conjugates. Error bars are standard deviations for = 3. As indicated by asterisk (*), only statistically significant differences were found between Pep2 and Pep11 in (A) using < 0.01). (C) Different locations of four representative peptides Pep 2, 5, 8, and 11 in an antibody, which results in different digestion kinetics in A. Free trypsin enzymes are relatively small, and they can access inner parts of the antibody layer for proteolysis. Thus, we proceeded to restrict proteolysis to the outmost surfaces of the bead conjugates. To achieve this, MEKK12 trypsin was immobilized onto MBs of the same size as the MB-Ab materials. In this way, the effective range of MB-trypsin is usually more strictly confined to the outermost regions of the antibody layer due to steric effects. MB-trypsin will have a difficult time reaching the inner regions of the Ab layer, so that if Abs are 100% oriented, only Fab peptides should be found by LC-MS/MS. Thus, MB-trypsin digestion of MB-ProA/G-Ab resulted in much larger digestion rates from peptides 2 and 5 from the Fab regions, compared to Fc-stem peptides 8 and 11 (Physique 5A). The proteolysis rates of MB-Ab conjugates are more compressed and comparable for the 4 peptides, even when a full 12 h reaction is usually observed (Physique S6)..