Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.

Poor understanding of the basic biology of Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis, hampers development of much-needed drugs, vaccines, and diagnostic tests. Better experimental tools are needed to expedite investigations of this pathogen at the systems level. Here, we present a functional MTB proteome microarray covering most of the proteome and an ORFome library. We demonstrate the broad applicability of the microarray by investigating global protein-protein interactions, small-molecule-protein binding, and serum biomarker discovery, identifying 59 PknG-interacting proteins, 30 bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding proteins, and 14 MTB proteins that together differentiate between tuberculosis (TB) patients with active disease and recovered individuals. Results suggest that the MTB rhamnose pathway is likely regulated by both the serine/threonine kinase PknG and c-di-GMP. This resource has the potential to generate a greater understanding of key biological processes in the pathogenesis of tuberculosis, possibly leading to more effective therapies for the treatment of this ancient disease.

Global identification of O-GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome.

O-Linked ß-N-acetylglucosamine (O-GlcNAcylation) is an important protein PTM, which is very abundant in mammalian cells. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT), whose substrate specificity is believed to be regulated through interactions with other proteins. There are a handful of known human OGT interactors, which is far from enough for fully elucidating the substrate specificity of OGT. To address this challenge, we used a human proteome microarray containing ~17,000 affinity-purified human proteins to globally identify OGT interactors and identified 25 OGT-binding proteins. Bioinformatics analysis showed that these interacting proteins play a variety of roles in a wide range of cellular functions and are highly enriched in intra-Golgi vesicle-mediated transport and vitamin biosynthetic processes. Combining newly identified OGT interactors with the interactors identified prior to this study, we have constructed the first OGT interactome. Bioinformatics analysis suggests that the OGT interactome plays important roles in protein transportation/localization and transcriptional regulation. The novel OGT interactors that we identified in this study could serve as a starting point for further functional analysis. Because of its high-throughput and parallel analysis capability, we strongly believe that protein microarrays could be easily applied for the global identification of regulators for other key enzymes.

Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

Fabrication and characterization of magnetic mesoporous silica nanospheres covalently bonded with europium complex.

A novel multifunctional nanocomposite has been developed by combining the magnetic (Fe3O4) cores encapsulated in the mesoporous silica nanospheres and the luminescent Eu(TTA)3phen complex covalently bonded to the framework of mesoporous silica through a chelate ligand 5-[N,N-bis-3-(triethoxysilyl)propyl]ureyl-1,10-phenanthroline (phen-Si). The obtained nanocomposite is denoted as Eu(TTA)3phen-MMS. It has been well characterized by field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), N2 adsorption/desorption, Quantum Design SQUID magnetometer and photoluminescence spectroscopy, respectively. The results demonstrated that Eu(TTA)3phen-MMS nanocomposite possess superparamagnetic behavior, intense red emission and mesostructures simultaneously.

Novel multifunctional nanocomposites: magnetic mesoporous silica nanospheres covalently bonded with near-infrared luminescent lanthanide complexes.

In this paper, we report the fabrication and characterization of magnetic mesoporous silica nanospheres covalently bonded with near-infrared (NIR) luminescent lanthanide complexes [denoted as Ln(DBM)(3)phen-MMS (Ln = Nd, Yb)]. Ln(DBM)(3)phen-MMS (Ln = Nd, Yb) nanospheres with an average size of 80-130 nm were synthesized via incorporation of the chelate ligand 5-[N,N-bis-3-(triethoxysilyl)propyl]ureyl-1,10-phenanthroline (phen-Si) into the framework of magnetic mesoporous silica (denoted as phen-MMS), followed by introduction of the Ln(DBM)(3)(H(2)O)(2) (Ln = Nd, Yb) complexes into the nanocomposites via a ligand exchange reaction. The morphological, structural, textural, magnetic, and NIR luminescent properties were well-characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), N(2) adsorption-desorption, a superconducting quantum interference device (SQUID), and photoluminescence spectra. These nanocomposites, which possess high surface area, high pore volume, and well-defined pore size, exhibit two-dimensional hexagonal (P6mm) mesostructures. After ligand-mediated excitation, Ln(DBM)(3)phen-MMS (Ln = Nd, Yb) nanocomposites exhibit the characteristic NIR emission of Nd(3+) and Yb(3+), respectively. Magnetic measurements reveal that these mulfunctional nanocomposites possess superparamagnetic properties at 300 K. The high magnetization values make the nanocomposites respond to the external magnetic field quickly. Additionally, the results indicate that Nd(DBM)(3)phen-MMS nanocomposites may have potential applications for laser systems or the optical amplifiers operating at 1.3 microm and Yb(DBM)(3)phen-MMS nanocomposites have several advantages for potential applications in drug delivery or optical imaging.