ID Enhancement and Age Selection {#s4d} ---------------------------------- The main purpose of this study was to develop an effective method for selection and purification of PICs to the extent of eliminating any other biological components with which the PICs may potentially be co-purified in vitro (i.e., a specific fraction of protein contaminants rather than as a part of any protein complex). Our experience has shown that it is critical to minimize nonspecific interactions of molecules with the magnetic beads, and therefore the optimal choice of buffers and ionic strength for antibody-coated, magnetically activated Dynabeads was the initial step toward achieving this goal. The second consideration was to develop a reliable and reproducible experimental design, and the third point was to minimize the labor involved in the initial set-up. To meet these goals, we used an empirical design with a maximum number of variables reduced to two and eliminated a potential problem by testing the antibody concentration at which the immunogen, IgG produced in rabbit, interacted with immunomagnetic beads. ### Magnetic Binding Capacity {#s4d1} Antibody-coated beads are extremely useful for the enrichment of high molecular weight protein complexes. However, their ability to bind proteins varies depending on the antibody concentration [@pone.0005520-Stepanova1], [@pone.0005520-Gentile2]. The binding capacity of Dynabeads for the proteins in the sample can be regulated by adjusting antibody concentration in the incubation buffer. One of the critical parameters for determining the correct volume of antibody required to saturate beads with protein is the size of the molecules interacting with the antibody. The binding capacity was lower when the bead to antibody ratio was 1∶2 (5 mg antibody per ml of Dynabeads) compared to a bead to antibody ratio of 1∶10 (1 mg antibody per ml of Dynabeads) or 1∶1 (500 µg of antibody per ml of Dynabeads). ### Binding of Protein Interaction Complexes {#s4d2} In our system, the Dynabeads were functionalized with affinity-purified rabbit antibody recognizing proteins from a whole-cell extract of DAPs. Our aim was to selectively bind proteins that bound to the antibody of the Dynabeads via noncovalent forces. The functionalized beads were incubated in a cell extract, which resulted in bead-protein complexes being formed. One of the characteristics of these complexes is that they were readily dissociated into their individual components by washing. This property is very desirable because it allowed us to analyze the composition of the DAPs via MS. The binding capacity of Dynabeads for DAPs, as analyzed by western blotting with MOMP antibodies, was in good agreement with the data obtained in mass spectrometry analysis of MOMP-interacting proteins. Several MOMP-specific antibodies that react with this molecule were successfully used for the purification of MOMP and for binding studies [@pone.0005520-Hobbs1], [@pone.0005520-Cao1], [@pone.0005520-Papadopoulos1], [@pone.0005520-Dintilhac1], [@pone.0005520-Chardin2], [@pone.0005520-Bergman1], [@pone.0005520-Bergman2]. However, other antibodies were completely ineffective for purification of MOMP. For example, the MOMP antiserum generated by immunization with a mixture of MOMP and recombinant proteins encoded by the genome of the GBS strain 2603V/R [@pone.0005520-Mikaelian1], [@pone.0005520-Mikaelian2] precipitated proteins other than MOMP [@pone.0005520-Gammon3], [@pone.0005520-Gammon4]. By contrast, a collection of anti-MOMP antibodies were used for purification and detection of MOMP by immunoprecipitation [@pone.0005520-Pannell1], [@pone.0005520-Zhu1], and showed that MOMP is conserved among pathogenic GBS strains [@pone.0005520-Achenbach2], [@pone.0005520-Dale1]. In our study, we used mouse monoclonal antibodies reacting with MOMP in ELISA. The results of the western blotting with these antibodies were ambiguous and did not match any other data obtained by mass spectrometry, suggesting the presence of a non-covalently bound form of MOMP. It is important to note that our experimental system provides for non-invasive analysis of interaction between DAPs and MOMP without inducing stress on either molecule. Also, these complexes were dissociated by a low ionic strength buffer. In summary, we described the use of Dynabeads coupled with an antibody against MOMP in immunoprecipitation of GBS MOMP-interacting proteins. Supporting Information {#s5} ====================== ###### Immunoprecipitation of proteins from GBS bacterial cells with antibodies against MOMP. The MOMP protein was first immunoprecipitated from the cell lysate using Dynabeads coupled to anti-MOMP antibodies (MOMP). The immunoprecipitate (MOMP-IP) and the starting lysate were subjected to SDS-PAGE, and were transferred to the nitrocellulose membrane. An anti-MOMP antibody raised against a full-length fragment of the protein was used for detection of MOMP on western blot. The presence of MOMP was confirmed in the immunoprecipitate but not in the original sample. We also observed a faint band corresponding to MOMP in the starting lysate. Since these bands were visible only in the gel stained with coomassie brilliant blue, these bands are not MOMP-specific. Although this experiment was reproduced more than ten times, the identity of the bands remained unclear. The result indicated the presence of noncovalent associations of proteins with MOMP. (0.33 MB TIF) ###### Click here for additional data file. ###### Antibody specificity of MOMP-interacting proteins. Immunoprecipitation was carried out with Dynabeads coupled with an anti-MOMP antibody. Proteins in the MOMP-IP were separated by 1-D SDS-PAGE and were electroblotted to PVDF membrane (A). Each gel lane was divided into three stripes, and each of them was subjected to western blot analysis with (1) antibody against MOMP (primary antibody), (2) antibody against MOMP (secondary antibody), and (3) antibody against EF-Tu. The proteins, EF-Tu and MOMP-binding proteins were visualized by western blotting with either primary or secondary antibodies, but not with the antibody against EF-Tu (C). By contrast, these proteins were absent in a control sample precipitated with Dynabeads coupled with an irrelevant antibody (B). The result indicated that only MOMP-binding proteins were present in the immunoprecipitate. (0.18 MB TIF) ###### Click here for additional data file. ###### Expression of MOMP-binding proteins in GBS cells. Total proteins (15 µg) from four GBS strains were separated by SDS-PAGE and were stained by Coomassie blue (A) or silver (B). MOMP-specific protein bands were identified by mass spectrometry (C). The results were obtained with three different cell lines: the protein band at ∼39 kDa was detected in the whole cell extracts of all the GBS strains tested and corresponds to the MOMP band identified by western blotting. Moreover, it corresponded to a specific GBS protein interacting with MOMP (data not shown). The protein bands observed at ∼42, ∼72, and ∼97 kDa in SDS-PAGE corresponded to the three GBS proteins interacting with MOMP that were identified by mass spectrometry. Note that the latter protein bands could be observed only in total proteins obtained from the culture. These results confirmed that at least some of the MOMP-binding proteins are translated and secreted by GBS. (0.43 MB TIF) ###### Click here for additional data file. We are grateful to the Core Facility for Proteomics at the University of Tennessee Health Science Center for providing their technical support with LC/MS/MS equipment. We thank Dr. J. Pannell and M.A. Borsos for the kind gift of plasmid pGEM4Z\_(pMOMP-FLAG) containing *pbp2a* gene. We thank Dr. I. V. Ermolenko for his comments regarding the project and A. Gorbunova for her helpful discussions. **Competing Interests:**The authors have declared that no competing interests exist. **Funding:**This work was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. [^1]: Conceived and designed the experiments: EAV JC LK SJ EIP. Performed the experiments: EAV JC. Analyzed the data: EAV JC SJ LK. Contributed reagents/materials/analysis tools: SJ EIP. Wrote the paper: EAV JC SJ EIP.