Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes

Abstract

Alpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen-independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic-based serum biomarker discovery.

Figure 1

A2M-substrate complexes immunoprecipitated from human serum. Immunoblot for A2M is shown. A2M exists in vivo as a tetramer of identical 185-kDa subunits. Under reducing SDS-PAGE conditions, non-substrate bound A2M monomers^b migrate at ~ 185 kDa. Proteolysis of the bait region leads to dissociation of the 185-kDa subunit into a 100-kDa subunit^d and an 85-kDa sub-unit not recognized by the antibody used in this experiment. Reactive cross linking of a substrate to a single 100-kDa subunit produces bands ranging from 120 to 180 kDa^c, depending on the size of the substrate protein. High molecular weight complexes (>250 kDa)^a represent multiple 100-kDa A2M subunits covalently bound to a single substrate. Immunoglobulin heavy chain (IgG HC) is shown. Lane 1: plasma; Lane 2: serum; Lane 3: A2M antibody only control. Lane 4: A2M immunoprecipitation from serum. Native and cross-linked A2M-substrate complexes are observed in both serum (Lane 2) and plasma (Lane 1) and can be immunoprecipitated (Lane 4) for analysis of associated substrate proteins.

Figure 2

Inter-sample reproducibility of protein identification by cohort. Overlap of detected proteins amongst all six patients from the cancer cohort (A) and the control cohort (B) is shown scaled to the number of peptides required for protein identification. The proportion of proteins detected in all six patients in both cohorts did not significantly vary as the number of required peptides for identification increased.

Figure 3

Measurement of HSP90α serum concentration. Serum was collected from an expanded cohort of cancer (n = 18) and control (n = 13) patients, and HSP90α concentration was measured by ELISA. The results are shown as a box and corresponding dot plot. Each solid dot represents an individual patient measurement. The horizontal bar transecting each box represents the median cohort value, and the boundaries of each box represent the upper and lower quartile values for each cohort. The bars extend to 1.5 times the inner quartile by convention. Outlying values are shown in the cancer cohort as hollow dots. There was an increased serum concentration of HSP90α in the cancer cohort relative to control (p = 0.001). The median concentration in cancer patients was 50.7 ng/mL (range 25.5–378.1), while the median for control subjects was 27.6 ng/mL (13.9–46.5). These data provide a separation of groups with an area under the ROC curve of 0.83 (95% confidence interval of 0.68 to 0.95).