Journal
THERANOSTICS
Volume 7, Issue 16, Pages 4057-4070Publisher
IVYSPRING INT PUBL
DOI: 10.7150/thno.20151
Keywords
Cell-free protein microarray; Proteomics; Protein-protein interaction; Antibody; Biomarker
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Funding
- National Natural Science Foundation of China [81673040]
- National International Cooperation Grant [2014DFB30010]
- State Key Laboratory of Proteomics [SKLP-O201504, SKLP-K201505]
- Beijing Municipal Human Resources and Social Security Bureau (Grant for Overseas Scholars)
- Guangdong Science and Technology Department [2016A020215004]
- Early Detection Research Network [5U01CA117374]
- National Institute of Allergy and Infectious Diseases (NIAID) [RO1AI096213]
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Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays. Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot. Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA. Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.
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