Antibody-array interaction mapping, a new method to detect protein complexes applied to the discovery and study of serum amyloid P interactions with kininogen in human plasma

Abstract

Protein-protein interactions are fundamentally important in biological processes, but the existing analytical tools have limited ability to sensitively and precisely measure the dynamic composition of protein complexes in biological samples. We report here the development of antibody-array interaction mapping (AAIM) to address that need. We used AAIM to probe interactions among a set of 48 proteins in serum and found several known interactions as well potentially novel interactions, including multiprotein clusters of interactions. A novel interaction initially identified between the innate immune system protein C-reactive protein and the inflammatory protein kininogen (KNG) was confirmed in subsequent experiments to involve serum amyloid P instead of its highly related family member, C-reactive protein. AAIM was used in a variety of formats to further study this interaction. In vitro studies confirmed the ability of the purified proteins to interact and revealed a zinc dependence of the interaction. Studies using plasma samples collected longitudinally following a controlled myocardial infarction revealed no consistent changes in the serum amyloid P-KNG interaction levels but consistent changes in KNG activation and interactions with plasma prekallikrein. These results demonstrate a versatile platform for measuring the dynamic composition of protein complexes in biological samples that should have value for studies of normal and disease-related signaling networks, multiprotein clusters, or enzymatic cascades.

Fig. 1.

Antibody-array interaction mapping. a, experimental plan. Using a microscope slide printed with 48 identical antibody microarrays, a single serum sample is incubated on all arrays. Each array is spotted with 48 different capture antibodies. After incubation and washing, each array is probed with one of 48 different detection antibodies, each of which corresponds to one of the capture antibodies. b, molecular detail. Each array is incubated with the same serum sample, resulting in the capture of hypothetical proteins 1, 2, and 3. The detection antibodies localize to the capture antibodies at which their targets are found. The location of the respective detection antibodies reveals information about potential interactions between the targeted proteins, in this case an interaction between proteins 1 and 3.

Fig. 2.

Probing interactions among set of serum proteins. Arrays were printed containing 48 different antibodies targeting various serum proteins. Each array was incubated with a pooled serum sample and probed with one of 48 different detection antibodies. a, scanned fluorescence from the entire slide. b, enlarged view of several arrays along with negative control arrays that were incubated with PBS instead of serum. A positive control spot, biotinylated BSA, was located in the lower right corner of each array. c, quantification of the fluorescence data. Each panel shows the fluorescence signal at the capture antibodies on one array. Ab, antibody; VEGF, vascular endothelial growth factor; PDGFb, platelet-derived growth factor subunit B.

Fig. 3.

Exploration of novel interaction. a, the antibodies and proteins used for probing this interaction. b, immunoprecipitation (IP) and Western blots. The immunoprecipitations were performed on pools of plasma samples from either cancer patients (lanes marked C) or healthy patients (lanes marked H). Both isoforms of kininogen, high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK), were immunoprecipitated using anti-kininogen antibodies because the heavy chain of kininogen targeted by that antibody is identical between the two isoforms. Lanes 7–10 are Western blots performed on separations of purified proteins. c, Western blot comparison of purified high molecular weight KNG and purified CRP, showing bands at 23–25 kDa that were detected with anti-CRP. d, Western blots detecting either purified CRP or SAP using various anti-CRP detection antibodies. The bands at ∼25 kDa are shown. hpCRP, human purified CRP; hSAP, human SAP; BK, bradykinin; NA, not applicable; HMW, high molecular weight.

Fig. 4.

In vitro studies of SAP-KNG and CRP-KNG interactions. a, KNG levels were measured using the indicated antibodies after the addition of CRP, SAP, or buffer to 1 μg/ml KNG in PBS supplemented with 2 mm CaCl₂. b, the same solutions as in a were probed for CRP levels at the CRP and KNG capture antibodies and at phosphocholine-linked BSA (PC-BSA) and for KNG levels at the CRP capture antibody. The experiments were run using either a calcium-supplemented buffer or a buffer supplemented with EDTA to remove trace calcium. Data are shown from a representative CRP capture antibody of 10 on the arrays. c, the same solutions were probed for SAP levels at the SAP and KNG capture antibodies and for KNG levels at the SAP captures. A pair of representative anti-SAP antibodies is presented of five on the arrays. d, SAP alone, KNG alone, or SAP plus KNG were incubated on arrays using buffers supplemented with various concentrations of zinc, which was added to PBS buffer as ZnCl₂. KNG was detected at SAP capture antibodies, and data from a representative antibody are shown. HMW, high molecular weight; NC, negative control.

Fig. 5.

Variation in interaction levels following acute phase responses. a, protein levels in longitudinally collected plasma samples from five patients undergoing induced myocardial infarction. CRP levels were measured by Western blot, and KNG, CRP, and SAP were measured by sandwich detection on antibody arrays. The plasma samples were diluted 200× in sample buffer for the antibody array analysis. Buffer indicates negative controls, which were arrays incubated with PBS buffer. b, the detection of protein complexes and activated KNG in longitudinal samples. KNG was detected at anti-SAP capture antibodies (left panel). A representative of two different anti-SAP antibodies is shown. KNG also was detected at an antibody specific for activated KNG (middle panel) and at an antibody specific for PPK (right panel). PK, plasma kallikrein.