Abstract
There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format.
Highlights
There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care
One of the advantages with mass spectrometry is that the method allows for the detection of differences in protein modifications, such as glycosylation or phosphorylation, which have been found useful for some applications [5]
Many potential biomarkers have been discovered using mass spectrometry, the approach is yet limited to the analysis of a relatively small number of patient samples
Summary
Antibodies—Protocols for antigen selection, cloning, expression, purification, and immunization of rabbits, followed by affinity purification to yield mono-specific polyclonal antibodies, and their characterization with Western blots and antigen microarrays were applied as described previously [20, 21]. The coupling efficiency for each antibody was determined via R-phycoerythrin-labeled anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories,) and a 100-plex bead mixture was created as described previously [23]. N-Hydroxysuccinimidyl ester of biotinoyl tetraoxapentadecanoic acid (Pierce) was added at 10-fold molar excess to yield an overall 1:10 sample dilution followed by a 2-h incubation at 4 °C in a microtiter plate shaker (Thermomixer, Eppendorf). All samples were subsequently utilized without removing unincorporated biotin and diluted 1:50 (Matrix PlateMate) in an assay buffer composed of 0.5% (w/v) polyvinyl alcohol and 0.8% (w/v) polyvinylpyrrolidone (Sigma) in 0.1% casein in PBS supplemented with 0.5 mg/ml rabbit IgG (Bethyl Laboratories). A limit of detection (LOD) was determined via a five-parametric logistic regression function to reveal the antigen concentration at 3ϫ standard deviation above a background mean, which was determined in a sample that had not been supplemented with antigen. The dependence between profiles and clinical PSA values was calculated using Spearman’s rank correlation ()
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