Abstract
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.
| Original language | English (US) |
|---|---|
| Article number | 20151 |
| Journal | Theranostics |
| Volume | 7 |
| Issue number | 16 |
| DOIs | |
| State | Published - 2017 |
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Keywords
- Antibody
- Biomarker
- Cell-free protein microarray
- Protein-protein interaction
- Proteomics
ASJC Scopus subject areas
- Medicine (miscellaneous)
- Pharmacology, Toxicology and Pharmaceutics (miscellaneous)
Cite this
Multiplexed Nucleic Acid programmable protein arrays. / Yu, Xiaobo; Song, Lusheng; Petritis, Brianne; Bian, Xiaofang; Wang, Haoyu; Viloria, Jennifer; Park, Jin; Bui, Hoang; Li, Han; Wang, Jie; Liu, Lei; Yang, Liuhui; Duan, Hu; McMurray, David N.; Achkar, Jacqueline M.; Magee, Dewey; Qiu, Ji; LaBaer, Joshua.
In: Theranostics, Vol. 7, No. 16, 20151, 2017.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Multiplexed Nucleic Acid programmable protein arrays
AU - Yu, Xiaobo
AU - Song, Lusheng
AU - Petritis, Brianne
AU - Bian, Xiaofang
AU - Wang, Haoyu
AU - Viloria, Jennifer
AU - Park, Jin
AU - Bui, Hoang
AU - Li, Han
AU - Wang, Jie
AU - Liu, Lei
AU - Yang, Liuhui
AU - Duan, Hu
AU - McMurray, David N.
AU - Achkar, Jacqueline M.
AU - Magee, Dewey
AU - Qiu, Ji
AU - LaBaer, Joshua
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Antibody
KW - Biomarker
KW - Cell-free protein microarray
KW - Protein-protein interaction
KW - Proteomics
UR - http://www.scopus.com/inward/record.url?scp=85030639126&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85030639126&partnerID=8YFLogxK
U2 - 10.7150/thno.20151
DO - 10.7150/thno.20151
M3 - Article
C2 - 29109798
AN - SCOPUS:85030639126
VL - 7
JO - Theranostics
JF - Theranostics
SN - 1838-7640
IS - 16
M1 - 20151
ER -