Identification of Sero-Diagnostic Antigens for the Early Diagnosis of Johne's Disease using MAP Protein Microarrays.

Considerable effort has been directed toward controlling Johne's disease (JD), a chronic granulomatous intestinal inflammatory disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) in cattle and other ruminants. However, progress in controlling the spread of MAP infection has been impeded by the lack of reliable diagnostic tests that can identify animals early in the infection process and help break the transmission chain. To identify reliable antigens for early diagnosis of MAP infection, we constructed a MAP protein array with 868 purified recombinant MAP proteins, and screened a total of 180 well-characterized serum samples from cows assigned to 4 groups based on previous serological and fecal test results: negative low exposure (NL, n = 30); negative high exposure (NH, n = 30); fecal-positive, ELISA-negative (F + E-, n = 60); and both fecal- and ELISA-positive (F + E+, n = 60). The analyses identified a total of 49 candidate antigens in the NH, F + E-, and F + E+ with reactivity compared with the NL group (p < 0.01), a majority of which have not been previously identified. While some of the antigens were identified as reactive in only one of the groups, others showed reactivity in multiple groups, including NH (n = 28), F + E- (n = 26), and F + E+ (n = 17) groups. Using combinations of top reactive antigens in each group, the results reveal sensitivities of 60.0%, 73.3%, and 81.7% in the NH, F + E-, and F + E+, respectively at 90% specificity, suggesting that early detection of infection in animals may be possible and enable better opportunities to reduce within herd transmission that may be otherwise missed by traditional serological assays that are biased towards more heavily infected animals. Together, the results suggest that several of the novel candidate antigens identified in this study, particularly those that were reactive in the NH and F + E- groups, have potential utility for the early sero-diagnosis of MAP infection.

Rapid immunodiagnostics of multiple viral infections in an acoustic microstreaming device with serum and saliva samples.

There is a growing need to screen multiple infections simultaneously rather than diagnosis of one pathogen at a time in order to improve the quality of healthcare while saving initial screening time and reduce costs. This is the first demonstration of a five-step protein array assay for the multiplexed detection of HIV, HPV and HSV antibodies on an integrated microfluidic system. HIV, HPV and HSV reactive antibodies from both serum and saliva were rapidly detected by acoustic streaming-based mixing and pumping to enable an integrated, rapid and simple-to-use multiplexed assay device. We validated this device with 37 serum and saliva samples to verify reactivity of patient antibodies with HIV, HPV and HSV antigens. Our technology can be adapted with different protein microarrays to detect a variety of other infections, thus demonstrating a powerful platform to detect multiple putative protein biomarkers for rapid detection of infectious diseases. This integrated microfluidic protein array platform is the basis of a potent strategy to delay progression of primary infection, reduce the risk of co-infections and prevent onward transmission of infections by point-of-care detection of multiple pathogens in both serum and oral fluids.

Biomimetic Design of Protein Nanomaterials for Hydrophobic Molecular Transport.

Biomaterials such as self-assembling biological complexes have demonstrated a variety of applications in materials science and nanotechnology. The functionality of protein-based materials, however, is often limited by the absence or locations of specific chemical conjugation sites. In this investigation, we developed a new strategy for loading organic molecules into the hollow cavity of a protein nanoparticle that relies only on non-covalent interactions, and we demonstrated its applicability in drug delivery. Based on a biomimetic model that incorporates multiple phenylalanines to create a generalized binding site, we retained and delivered the antitumor compound doxorubicin by redesigning a caged protein scaffold. Through an iterative combination of structural modeling and protein engineering, we obtained new variants of the E2 subunit of pyruvate dehydrogenase with varying levels of drug-carrying capabilities. We found that an increasing number of introduced phenylalanines within the scaffold cavity generally resulted in greater drug loading capacities. Drug loading levels could be achieved that were greater than conventional nanoparticle delivery systems. These protein nanoparticles containing doxorubicin were taken up by breast cancer cells and induced significant cell death. Our novel approach demonstrates a universal strategy to design de novo hydrophobic binding domains within protein-based scaffolds for molecular encapsulation and transport, and it broadens the ability to attach guest molecules to this class of materials.

Development of a novel cross-linking strategy for fast and accurate identification of cross-linked peptides of protein complexes.

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.