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.

O-Linked β-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.