Summary Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly) containing isopeptides following trypsin digestion. We identify ~19,000 diGly modified lysine residues within ~ 5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition indicating both a dependence of on-going translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.

Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and proteinprotein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases. R ecent years have witnessed significant interest in biological applications of novel inorganic nanomaterials such as nanocrystals (1, 2), nanowires (3), and nanotubes (4, 5) with the motivation to create new types of analytical tools for life science and biotechnology. Single-walled carbon nanotubes (SWNTs) are interesting molecular wires (diameter Ϸ1-2 nm) with unique electronic properties that have been spotlighted for future solid-state nanoelectronics (6, 7). Bridging nanotubes with biological systems, however, is a relatively unexplored area, with the exception of a few reports on nanotube probe tips for biological imaging (4), nonspecific binding (NSB) of proteins (8-10), functionalization chemistry for bioimmobilization on nanotube sidewalls (5), and one study on biocompatibility (11).Previously, we and others have shown that the electrical conductance of a nanotube is highly sensitive to its environment and varies significantly with changes in electrostatic charges and surface adsorption of various molecules (12)(13)(14). This research has hinted at possible SWNT-based miniature sensors for detecting biological molecules in fluids. Here, we systematically explore how nanotubes interact with and respond to various proteins in solution, how chemical functionalization can be used to tailor these interactions, and how the resulting understanding enables highly selective nanotube sensors for the electronic detection of proteins. Using atomic force microscopy (AFM) and quartz crystal microbalance (QCM) and electronic transport measurements, we first reveal that proteins in general exhibit a high degree of NSB on nanotubes, a phenomenon undesirable for potential biosensors. We then demonstrate a functionalization scheme involving irreversible adsorption of Tween 20 or triblock copolymer chains on nanotubes to prevent this general NSB, while at the same time enabling the binding of specific proteins of interest that can be detected electronically without the need for labeling. Further, we demonstrate specific detection of mAbs to the human autoantigen U1A, a prototype target of the autoimmune response in patients with systemic lupu...

Carbon nanotube field-effect transistors commonly comprise nanotubes lying on SiO 2 surfaces exposed to the ambient environment. It is shown here that the transistors exhibit hysteresis in their electrical characteristics because of charge trapping by water molecules around the nanotubes, including SiO 2 surface-bound water proximal to the nanotubes. Hysteresis persists for the transistors in vacuum since the SiO 2bound water does not completely desorb in vacuum at room temperature, a known phenomenon in SiO 2 surface chemistry. Heating under dry conditions significantly removes water and reduces hysteresis in the transistors. Nearly hysteresis-free transistors are obtainable by passivating the devices with polymers that hydrogen bond with silanol groups on SiO 2 (e.g., with poly(methyl methacrylate) (PMMA)). However, nanotube humidity sensors could be explored with suitable water-sensitive coatings. The results may have implications to field-effect transistors made from other chemically derived materials.

Nanotechnology has received increased attention in the biological research field. The important examples are (1) the usage of nanoparticles in optical and magnetic resonance imaging; 1,2 (2) the demonstration of potential application of metal nanoshells and carbon nanotubes for the treatment of tumor and cancer cells; 3,4 and (3) the application of nanowire-based transistors to electrically detect specific biomolecules. 5,6 In all of these cases, the nanomaterials are functioning either inside the cells or at the vicinity of the surface of biomolecules. Direct interconnection of the cells to the external world by interfacing nanomaterials may afford great opportunities to probe and manipulate biological processes occurring inside the cells, across the membranes, and between neighboring cells. 7,8 A nanoscale material with high aspect ratio is a good candidate for this application. For instance, silicon nanowires (SiNWs, d ) 1-100 nm) are a few orders of magnitude smaller in diameter than mammalian cells (d cell ∼ on the order of 10 µm) yet comparable to the sizes of various intracellular biomolecules. The nanowires have high aspect ratio (<10 3 ) and yet are sufficiently rigid to be mechanically manipulated. The nanometer scale diameter and the high aspect ratio of SiNWs make them readily accessible to the interiors of living cells, which may facilitate the study of the complex regulatory and signaling patterns at the molecular level.In this Communication, we present the first demonstration of a direct interface of silicon nanowires with mammalian cells such as mouse embryonic stem (mES) cells and human embryonic kidney (HEK 293T) cells without any external force. The cells were cultured on a silicon (Si) substrate with a vertically aligned SiNW array on it. The penetration of the SiNW array into individual cells naturally occurred during the cell incubation. The cells survived up to several days on the nanowire substrates. The longevity of the cells was highly dependent on the diameter of SiNWs. Furthermore, successful maintenance of cardiac myocytes derived from mES cells on the wire array substrates was observed, and gene delivery using the SiNW array was demonstrated.SiNWs were synthesized vertically aligned with respect to Si-(111) substrates via chemical vapor deposition as described earlier. 9 The diameter of the nanowires was controlled by the size of gold nanoparticles that were used as catalytic seeds for the nanowire synthesis or by reducing the diameter of Si nanowires via oxidation and subsequent hydrofluoric (HF) acid etching step. 10 The SiNW substrates had a native oxide layer and were used without any surface modification unless otherwise specified. Before any exposure to living cells, the substrates were sterilized in a solvent of 70% ethanol and 30% sterile water.First, physical interaction between the nanowires and the cells was studied using confocal microscopy and scanning electron microscopy (SEM). Mouse embryonic stem cells stably expressing green fluorescent protein (GFP) were cultured o...