NAPPA Technology

Overview   |   Applications   |   Publications

Overview


 

The foundation of our proteomics research is a novel protein microarray technology, called Nucleic Acid-Programmable Protein Array (NAPPA), which replaces the complex process of spotting purified proteins with the simple process of spotting plasmid DNA. This core technology is a key innovation in the quest for identifying and characterizing proteins that regulate crucial events in the progression of diseases such as type I diabetesbreast and lung cancer, or infectious pathogens that are currently the focus of our research.

Protein microarrays display proteins in high spatial density on a microscopic surface.  They can be used to test a variety of functions of many proteins simultaneously including interactions with other macromolecules, functional activity, and their suitability as substrates of enzymes.  Historically, these arrays have been produced by expressing and purifying proteins, which are then printed on the array surface.  However, many challenges accompany this approach including the difficulty in purifying many proteins, a dynamic range in yields that spans several orders of magnitude (which is reflected in the widely varying amount of protein displayed on the array surface) and the danger or proteins becoming unfolded during the many manipulations, such as purification, storage and printing.

To avoid these challenges and to produce the most functional arrays possible, we have developed a novel protein microarray technology, called Nucleic Acid-Programmable Protein Array (NAPPA), which replaces the complex process of spotting purified proteins with the simple process of spotting plasmid DNA. Our technology exploits the ability to transfer protein encoding regions (open reading frames; ORFs) into specialized tagged expression vectors. These new expression clones are then spotted on the array and the proteins are then produced in situ in a cell-free system and immobilized in place upon their synthesis. This minimizes direct manipulation of the proteins and produces them just-in-time for the experiment, avoiding problems with protein purification and stability (Science. 2004 305:86). A next generation method for these arrays has been developed that allows thousands of proteins to be produced simultaneously in situ, and with remarkably consistent protein levels displayed (Ramachandran et al 2008). More recently, our development has focused on expressing protein using human HeLa lysate (Festa et al 2012), on changing the tags on the proteins to be able to display them in denaturing environments to detect linear epitopes (Wang et al 2012), and on increasing the density of the spots with the goal of printing the entire human proteome on one array (Takulapalli et al 2012).

In addition to our center's research, we now offer NAPPA array services as part of our NAPPA Protein Array Core facility. You can learn more about it here or on our NAPPA Core Website.

 

 

 

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Applications

The power of NAPPA is that by expressing many proteins on a single array, it is possible to test the function of many proteins simultaneously.  In our laboratory we use NAPPA technology to explore numerous biological questions including:

  1. Identifying autoantibody biomarkers in sera that can be readily used for the detection of cancer, such as breast cancer, ovarian cancer, prostate cancer, HPV-positive cancers and lung cancer.
  2. Identifying novel autoantibodies for diagnosis of Type I diabetes, inflammatory bowel disease, POEMS syndrome
  3. Developing a better vaccine against B. anthracis.
  4. Screening human serum in order to find antibodies against specific pathogens such as Pseudomonas aeruginosa, Chlorea, Tuberculosis to identify immunogenic proteins as a first step towards developing an effective vaccine.
  5. Detect post-translational modifications such as phosphorylation, AMPylation, or citrullination
  6. Identify protein-protein or protein-DNA interactions
  7. Quantify protein binding kinetics in high throughput by coupling NAPPA with surface plasmon resonance (SPRi) to identify affinity reagent binding or kinetics of the B-cell pathway

 


 

Publications

Biological Applications

Miersch S, Bian X, Wallstrom G, Sibani S, Logvinenko T, Wasserfall CH, Schatz D, Atkinson M, Qiu J, Labaer J. (2013) Serological autoantibody profiling of type 1 diabetes by protein arrays. J Proteomics. 2013 Oct 19. pii: S1874-3919(13)00530-7. doi: 10.1016/j.jprot.2013.10.018. [Epub ahead of print] Abstract

Xiaobo Yu, Garrick Wallstrom, Dewey Mitchell Magee, Ji Qiu, D. Eliseo A. Mendoza, Jie Wang, Xiaofang Bian, Morgan Graves, and Joshua LaBaer. Quantifying antibody binding on protein microarrays using microarray nonlinear calibration. Biotechniques. 2013 May;54(5):257-64. doi: 10.2144/000114028. Abstract

Wallstrom G, Anderson KS, and LaBaer J.  Biomarker Discovery for Heterogeneous Diseases.  Cancer Epidemiology Biomarkers & Prevention.  In Press.  Accepted Feb 26, 2013. Abstract

Manzano-Roman R, Diaz-Martin V, Gonzalez-Gonzalez M, Matarraz S, Alvarez-Prado AF, Labaer J, Orfao A, Perez-Sanchez R, Fuentes M. Self-assembled protein arrays from an ornithodos moubata salivary gland expression library. J Proteome Res 2012/11/13. doi: 10.1021/pr300696h/ [Epub ahead of print] Abstract

Gibson DS, Qiu J, Mendoza EA, Barker K, Rooney ME, LaBaer J. Circulating and synovial antibody profiling of juvenile arthritis patients by nucleic acid programmable protein arrays. Arthritis Res Ther. Apr 17;14(2):R77. [Epub ahead of print] Abstract

Wright C, Sibani S, Trudgian D, Fischer R, Kessler B, LaBaer J, Bowness P. Detection of multiple autoantibodies in patients with ankylosing spondylitis using nucleic Acid programmable protein arrays. Mol Cell Proteomics. 11 (2), M9.00384. Abstract

Anderson KS, Sibani S, Wallstrom G, Qiu J, Mendoza EA, Raphael J, Hainsworth E, Montor WR, Wong J, Park JG, Lokko N, Logvinenko T, Ramachandran N, Godwin AK, Marks J, Engstrom P, Labaer J. (2011) Protein Microarray Signature of Autoantibody Biomarkers for the Early Detection of Breast Cancer. J Proteome Res Jan 7;10(1):85-96. Epub 2010 Nov 23. Abstract

Anderson KS, Wong J, D'Souza G, Riemer AB, Lorch J, Haddad R, Pai SI, Longtine J, McClean M, LaBaer J, Kelsey KT, Posner M. (2011) Serum antibodies to the HPV16 proteome as biomarkers for head and neck cancer. Br J Cancer Jun 7;104(12):1896-905. Abstract

Ceroni A, Sibani S, Baiker A, Pothineni VR, Bailer SM, LaBaer J, Haas J, Campbell CJ. (2010) Systematic analysis of the IgG antibody immune response against varicella zoster virus (VZV) using a self-assembled protein microarray. Mol Biosyst Sep;6(9):1604-10. Epub 2010 Jun 1. Abstract

Montor WR, Huang J, Hu Y, Hainsworth E, Lynch S, Kronish JW, Ordonez CL, Logvinenko T, Lory S, LaBaer J. (2009) Genome-wide study of Pseudomonas aeruginosa outer membrane protein immunogenicity using self-assembling protein microarrays. Infection and Immunity Nov;77(11):4877-86. Epub 2009 Sep 8. Abstract

Anderson KS, Ramachandran N, Wong J, Raphael JV, Hainsworth E, Demirkan G, Cramer D, Aronzon D, Hodi FS, Harris L, Logvinenko T, LaBaer J. (2008) Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer. J Proteome Res. 7(4):1490-9. Epub 2008 Feb 27. Abstract

Ramachandran N, Anderson KS, Raphael JV, Hainsworth E, Sibani S, Montor WR, Pacek M, Wong J, Elianne M, Sanda MG, Hu Y, Lovinenko T, Labaer J. (2008) Tracking humoral responses using self assembling protein microarrays. Proteomics Clin. Appl. (10/11): 1518-1527. Abstract

 

 


NAPPA Methods and Technology Development

Wang J, Barker K, Steel J, Park J, Saul J, Festa F, Wallstrom G, Yu X, Bian X, Anderson KS, Figueroa J, Labaer J, Qiu J. A versatile protein microarray platform enabling antibody profiling against denatured proteinsProteomics Clin Appl. 2012 Oct 2. doi: 10.1002/prca.201200062. [Epub ahead of print] Abstract

Festa F, Rollins SM, Vatten K, Hathaway M, Lorenz P, Mendoza EA, Yu X, Qui J, Kilmer G, Jensen P, Webb B, Ryan ET, LaBaer J - Robust microarray production of freshly expressed proteins in a human milieu. Proteomics Clin Appl. 2012 Oct 2. doi: 10.1002/prca.201200063. [Epub ahead of print] PMID: 23027544 Abstract

Takulapalli BR, Qiu J, Magee DM, Kahn P, Brunner A, Barker K, Means S, Miersch S, Bian X, Mendoza A, Festa F, Syal K, Park JG, LaBaer J, and Wiktor P. High Density Diffusion-Free Nanowell Arrays. Journal of Proteome Research,  2012 Aug 3;11(8):4382-91. Epub 2012 Jul 13.  PMID: 22742968 dx.doi.org/10.1021/pr300467q Abstract

Miersch S, and Labaer J. (2011) Nucleic Acid programmable protein arrays: versatile tools for array-based functional protein studies. Curr Protoc Protein Sci. Apr;Chapter 27:Unit27.2. Abstract

Qiu J, Labaer J. (2011) Nucleic Acid programmable protein array a just-in-time multiplexed protein expression and purification platform. Methods Enzymolgy 500:151-63. Abstract

Sibani S, and Labaer J. (2011) Immunoprofiling Using NAPPA Protein Microarrays. Methods Mol Biol. 723:149-61. Abstract

Wong, J., Sibani, S., Lokko, N. N., LaBaer, J., and Anderson, K. S. (2009) Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays. Journal of Immunological Methods 350, (1-2), 171-182. Abstract

Ramachandran N, Raphael JV, Hainsworth E, Demirkan G, Fuentes MG, Rolfs A, Hu Y, Labaer J. (2008) Next-generation high-density self-assembling functional protein arrays. NatMethods (5):535.

Ramachandran N, Srivastava S, Labaer J. (2008) Applications of protein microarrays for biomarker discovery. Proteomics Clin. Appl. (10/11): 1444-1459. Abstract

Ramachandran N, Hainsworth E, Demirkan G, LaBaer J.(2006) On-chip protein synthesis for making microarrays. Methods Mol Biol. 2006;328:1-14. Abstract

 



Reviews and Book Chapters

Lee, J. R.; Magee, D. M.; Gaster, R. S.; LaBaer, J.; Wang, S. X., Emerging protein array technologies for proteomics. Expert review of proteomics 2013, 10, (1), 65-75. Abstract

Qiu, J. and Anderson, KS, Proteomic and Metabolomic Approaches to Biomarker Discovery, Autoantibodies and Biomarker Discovery, Editors Haleem Issaq and Tim Veenstra,  Academic Press: 2012 

Ramachandran N, Srivastava S, Labaer J. (2008) Applications of protein microarrays for biomarker discovery. Proteomics Clin. Appl. (10/11): 1444-1459.