Automation of On-Resin Enrichment of S-Nitrosylated Proteins for Oxidized Cysteine-Selective cPILOT.

S-Nitrosylation (SNO) is a cysteine post-translational modification that increases with normal aging and is present in Alzheimer's disease and other aging-related illnesses. Detection of SNO-modified proteins can be challenging; however, we previously developed a robust quantitative proteomics approach termed "Oxidized Cysteine-Selective combined precursor isobaric labeling and isobaric tagging (OxcyscPILOT)" that allows for detection of endogenous SNO-modified proteins. OxcyscPILOT involves enrichment of SNO-modified proteins using a thiol-based resin. This enrichment is performed manually, and wash steps with the resin require numerous stages and buffer reagents. The goal of this study is to transfer the manual protocol to an automated liquid handler system in order to reduce wash steps, increase sample throughput, and minimize experimental error. In order to accomplish this, we evaluated the Biomek i7 liquid handler automated workstation and a Positive Pressure ALP (PPA) apparatus to conduct automated on-resin enrichment. Our findings provide starting pressure conditions for the use of PPA in an automated OxcyscPILOT proteomics workflow that could be transferred to other robotic liquid handling systems.

Quantitative proteomics to study aging in rabbit liver.

Aging globally effects cellular and organismal metabolism across a range of mammalian species, including humans and rabbits. Rabbits (Oryctolagus cuniculus are an attractive model system of aging due to their genetic similarity with humans and their short lifespans. This model can be used to understand metabolic changes in aging especially in major organs such as liver where we detected pronounced variations in fat metabolism, mitochondrial dysfunction, and protein degradation. Such changes in the liver are consistent across several mammalian species however in rabbits the downstream effects of these changes have not yet been explored. We have applied proteomics to study changes in the liver proteins from young, middle, and old age rabbits using a multiplexing cPILOT strategy. This resulted in the identification of 2,586 liver proteins, among which 45 proteins had significant p < 0.05) changes with aging. Seven proteins were differentially-expressed at all ages and include fatty acid binding protein, aldehyde dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl CoA dehydrogenase, apolipoprotein C3, peroxisomal sarcosine oxidase, adhesion G-protein coupled receptor, and glutamate ionotropic receptor kinate. Insights to how alterations in metabolism affect protein expression in liver have been gained and demonstrate the utility of rabbit as a model of aging.

The roles of S-nitrosylation and S-glutathionylation in Alzheimer's disease.

Alzheimer's disease (AD) is a debilitating dementia with complex pathophysiological alterations including modifications to endogenous cysteine. S-nitrosylation (SNO) is a well-studied posttranslational modification (PTM) in the context of AD while S-glutathionylation (PSSG) remains less studied. Excess reactive oxygen and reactive nitrogen species (ROS/RNS) directly or indirectly generate SNO and PSSG. SNO is dysregulated in AD and plays a pervasive role in processes such as protein function, cell signaling, metabolism, and apoptosis. Despite some studies into the role of SNO in AD, multiple identified SNO proteins lack deep investigation and SNO modifications outside of brain tissues are limited, leaving the full role of SNO in AD to be elucidated. PSSG homeostasis is perturbed in AD and may affect a myriad of cellular processes. Here we overview the role of nitric oxide (NO) in AD, discuss proteomic methodologies to investigate SNO and PSSG, and review SNO and PSSG in AD. A more thorough understanding of SNO, PSSG, and other cysteinyl PTMs in AD will be helpful for the development of novel therapeutics against neurodegenerative diseases.

3D-templated, fully automated microfluidic input/output multiplexer for endocrine tissue culture and secretion sampling.

A fully automated, 16-channel microfluidic input/output multiplexer (µMUX) has been developed for interfacing to primary cells and to improve understanding of the dynamics of endocrine tissue function. The device utilizes pressure driven push-up valves for precise manipulation of nutrient input and hormone output dynamics, allowing time resolved interrogation of the cells. The ability to alternate any of the 16 channels from input to output, and vice versa, provides for high experimental flexibility without the need to alter microchannel designs. 3D-printed interface templates were custom designed to sculpt the above-channel polydimethylsiloxane (PDMS) in microdevices, creating millimeter scale reservoirs and confinement chambers to interface primary murine islets and adipose tissue explants to the µMUX sampling channels. This µMUX device and control system was first programmed for dynamic studies of pancreatic islet function to collect ∼90 minute insulin secretion profiles from groups of ∼10 islets. The automated system was also operated in temporal stimulation and cell imaging mode. Adipose tissue explants were exposed to a temporal mimic of post-prandial insulin and glucose levels, while simultaneous switching between labeled and unlabeled free fatty acid permitted fluorescent imaging of fatty acid uptake dynamics in real time over a ∼2.5 hour period. Application with varying stimulation and sampling modes on multiple murine tissue types highlights the inherent flexibility of this novel, 3D-templated µMUX device. The tissue culture reservoirs and µMUX control components presented herein should be adaptable as individual modules in other microfluidic systems, such as organ-on-a-chip devices, and should be translatable to different tissues such as liver, heart, skeletal muscle, and others.

Macro-to-micro interfacing to microfluidic channels using 3D-printed templates: application to time-resolved secretion sampling of endocrine tissue.

Employing 3D-printed templates for macro-to-micro interfacing, a passively operated polydimethysiloxane (PDMS) microfluidic device was designed for time-resolved secretion sampling from primary murine islets and epidiymal white adipose tissue explants. Interfacing in similar devices is typically accomplished through manually punched or drilled fluidic reservoirs. We previously introduced the concept of using hand fabricated polymer inserts to template cell culture and sampling reservoirs into PDMS devices, allowing rapid stimulation and sampling of endocrine tissue. However, fabrication of the fluidic reservoirs was time consuming, tedious, and was prone to errors during device curing. Here, we have implemented computer-aided design and 3D printing to circumvent these fabrication obstacles. In addition to rapid prototyping and design iteration advantages, the ability to match these 3D-printed interface templates with channel patterns is highly beneficial. By digitizing the template fabrication process, more robust components can be produced with reduced fabrication variability. Herein, 3D-printed templates were used for sculpting millimetre-scale reservoirs into the above-channel, bulk PDMS in passively-operated, eight-channel devices designed for time-resolved secretion sampling of murine tissue. Devices were proven functional by temporally assaying glucose-stimulated insulin secretion from <10 pancreatic islets and glycerol secretion from 2 mm adipose tissue explants, suggesting that 3D-printed interface templates could be applicable to a variety of cells and tissue types. More generally, this work validates desktop 3D printers as versatile interfacing tools in microfluidic laboratories.