Journal
PROTEOMICS
Volume 11, Issue 23, Pages 4578-4582Publisher
WILEY
DOI: 10.1002/pmic.201000744
Keywords
Antibody microarrays; Chromatographic enrichment; Fluorescent labeling; Multiplexed proteomics technology; Protein arrays
Funding
- Norwegian Research Council (FUGE)
- University of Oslo
- Norwegian Cancer Society
- Norwegian Financial Mechanism [A/CZ0046/2/0008]
- EEA Financial Mechanism
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Antibody array analysis of complex samples requires capture reagents with exceptional specificity. The frequency of antibodies with label-based detection may be as low as 5%. Here, however, we show that as many as 25% of commercially available antibodies are useful when biotinylated cellular proteins are fractionated by size exclusion chromatography (SEC) first. A microsphere multiplex with 1725 antibodies to cellular proteins was added to 24 SEC fractions, labelled with streptavidin and analyzed by flow cytometry (microsphere-based affinity proteomics, MAP) The SEC-MAP approach resolved different targets captured by each antibody as reactivity peaks across the separation range of the SEC column (10-670kDa). Complex reactivity profiles demonstrated that most antibodies bound more than one target. However, specific binding was readily detected as reactivity peaks common for different antibodies to the same protein. We optimized sample preparation and found that amine-reactive biotin rarely inhibited antibody binding when the biotin to lysine ratio was kept below 1: 1 during labelling. Moreover, several epitopes that were inaccessible to antibodies in native proteins were unmasked after heat denaturation with 0.1% of SDS. The SEC-MAP format should allow researchers to build multiplexed assays with antibodies purchased for use in e. g. Western blotting.
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