4. Recent Technical Advances to the Platform
Several developments in protein array technology have allowed an improvement in throughput and sensitivity, achieving better capture and probing of proteins.
For instance, Wang and colleagues have described the use of human cell-free lysates (HeLa cell lysate as an in vitro transcription/translation system) in NAPPA arrays for protein expression enhancing protein yield and for presenting both natural and denatured forms of proteins for antibody biomarker discovery. Through their results, they demonstrated that autoantibody profiles from denatured protein arrays were distinct from those native protein arrays when probing with plasma samples. Furthermore, they blocked the protein arrays with E. coli lysates, reducing the background and improving the antibody signals [27]. Another study from the same lab reported an improvement in protein display by using the human cell-free lysate (10-fold higher) compared to the conventional rabbit reticulocyte lysate [28]. In turn, Xiabo and collaborators used NAPPA technology for the detection of global pathogen-host AMPylation (adenylytation PTM). Specifically, they developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays for the screening of 10,000 unique human proteins with Vibrio parahaemolyticus and Histophilus somni, identifying new AMPylation substrates (including Rac 2 and Rac 3) [29].
Collaboration between the Nicolini and LaBaer groups reported an innovative kind of NAPPA platform, in which the cDNA includes the SNAP tag and the expression is performed by using the PURE system (reconstituted from the purified components necessary for E. coli translation). Their principal purpose was to achieve the combination of mass spectrometry and fluorescence technology for protein microarrays. Their results with the PURE system showed a protein yield about 20 times higher with respect to the rabbit reticulocyte expression system [30].
Additionally, LaBaer’s lab has developed a very high density NAPPA array without any diffusion or contamination between spots by depositing the samples on 8000 nano-volume wells and, recently, the protein expression lysate into each well to achieve the protein expression in a HT manner. Furthermore, they present preliminary results with an ultra-high density protein array including up to 24,000 nanowells [31].
In turn, Nicolini and collaborators demonstrated the effective use of label-free approaches (anodic porous alumina, APA; and atomic force microscopy, AFM) in combination with NAPPA technology to test the expression and the atomic structure of proteins of interest. These studies show the possibility of overcoming limitations at the fluorescence detection level [32].
microarrays-04-00214-t001_Table 1 Table 1  Summary of NAPPA protein microarray applications.   TMZ, temozolomide; NAPPA, Nucleic Acid Programmable Protein Array; VZV, varicella zoster virus; APA, anodic porous alumina; AFM, atomic force microscopy.

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