Two-dimensional nitrosylated protein fingerprinting by using poly (methyl methacrylate) microchips

Abstract

S-nitrosylation (also referred to as nitrosation), a reversible post translational modification (PTM) of cysteine, plays an important role in cellular functions and cell signalling pathways. Nitrosylated proteins are considered as biomarkers of aging and Alzheimer's disease (AD). Microfluidics has been widely used for development of novel tools for separation of protein mixtures. Here we demonstrate two-dimensional micro-electrophoresis (2D μ-CE) separations of nitrosylated proteins from the human colon epithelial adenocarcinoma cells (HT-29) and AD transgenic mice brain tissues. Sodium dodecyl sulphate micro-capillary gel electrophoresis (SDS μ-CGE) and microemulsion electrokinetic chromatography (MEEKC) were used for the first and second dimensional separations, respectively. The effective separation lengths for both dimensions were 10 mm, and electrokinetic injection was used with field strength at 200 V cm⁻¹. After 80 s separation in the first CGE dimension, fractions were successfully transferred to a second MEEKC dimension for a short 10 s separation. We first demonstrate this 2D μ-CE separation by resolving five standard proteins with molecular weight (MW) ranging from 20 to 64 kDa. We also present a high peak capacity 3D landscape image of nitrosylated proteins from HT-29 cells before and following menadione (MQ) treatment to induce oxidative stress. Additionally, to illustrate the potential of the 2D μ-CE separation method for rapid profiling of oxidative stress-induced biomarkers implicated in AD disease, the nitrosylated protein fingerprints from 11-month-old AD transgenic mice brain and their age matched controls were also generated. To our knowledge, this is the first report on 2D profiling of nitrosylated proteins in biological samples on a microchip. The characteristics of this biomarker profiling will potentially serve as the screening for early detection of AD.