Generating high peak capacity 2-D maps of complex proteomes using PMMA microchip electrophoresis

A high peak capacity 2-D protein separation system combining SDS micro-CGE (SDS μ-CGE) with microchip MEKC (μ-MEKC) using a PMMA microfluidic is reported. The utility of the 2-D microchip was demonstrated by generating a 2-D map from a complex biological sample containing a large number of constituent proteins using fetal calf serum (FCS) as the model system. The proteins were labeled with a thiol-reactive AlexaFluor 633 fluorophore (excitation/emission: 633/652 nm) to allow for ultra-sensitive on-chip detection using LIF following the 2-D separation. The high-resolution separation of the proteins was accomplished based on their size in the SDS μ-CGE dimension and their interaction with micelles in the μ-MEKC dimension. A comprehensive 2-D SDS μ-CGE x μ-MEKC separation of the FCS proteins was completed inf less than <30 min using this 2-D microchip format, which consisted of 60 mm and 50 mm effective separation lengths for the first and second separation dimensions, respectively. Results obtained from the microchip separation were compared with protein maps acquired using conventional 2-D IEF and SDS-PAGE of a similar FCS sample. The microchip 2-D separation was found to be ∼60 x faster and yielded an average peak capacity of 2600 (±149), nearly three times larger than that obtained using conventional IEF/SDS-PAGE.