Challenges in High Resolution Multi-dimensional Electrophoresis for Proteomics: From the Macro- to Micro-Scale

John K. Osiri1, 2 and Steven A. Soper1, 2, 3
1Department of Chemistry and 2Center for BioModular Multi-Scale Systems,
3Department of Mechanical Engineering, Louisiana State University,
Louisiana State University, Baton Rouge, Louisiana 70803

For more information on this and other projects, see the Soper Research Group website

Significant efforts are being invested into developing new and innovative technologies directed toward handling large-scale proteomic-based discovery projects for generating new biomarkers that can be used for the diagnosis and prognosis of various diseases. Due to the complexity of most proteomes in terms of the enormous number of different components required to be analyzed, high demands have been placed on the associated technology platforms. As an example, the mammalian serum proteome contains an estimated number of different protein components exceeding 10,550,000.1 Therefore, top-down or bottom-up proteomics in many cases requires as the first stage in the processing pipeline, the ability to separate intact proteins using multi-dimensional electrophoresis. The multi-dimensional format is typically adopted because it can generate high peak capacities. Multi-dimensional separations utilize a format in which orthogonal separation mechanisms are used to sequentially sort the components contained within the sample. The work horse of most multi-dimensional separations for proteomics has been based on two-dimensional electrophoresis using iso-electric focusing (IEF) in the first dimension, which sorts proteins according to their iso-electric points, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which sorts the proteins according to differences in their molecular weights. In most cases, this separation is performed in a macro-scale version (~22 cm length gels) due to its relatively high peak capacity generated by the high degree of orthogonality between the individual separation dimensions and also, the long separation lengths producing high plate numbers. However, microchip electrophoresis platforms are viewed as attractive alternatives to their macro-scale counterparts in spite of their shorter separation lengths.2,3 One of the attractive aspects of these planar chip formats is the ability to integrate other processing steps onto the chip to reduce operator sample handling.4 Examples from the literature have demonstrated the ability to integrate several of the proteomic processing steps onto a chip. Foote and co-workers fabricated a microdevice that electrophoretically pre-concentrated fluorescently-labeled proteins prior to SDS micro-capillary gel electrophoresis (µ-CGE) using a porous silica membrane as the pre-concentrator.5 Dahlin and co-workers fabricated a poly(dimethylsiloxane) microchip in which six-peptide mixtures, dissolved in a physiological salt solution, were desalted, separated, and sprayed into a mass spectrometer for identification via peptide mass fingerprinting.6 Figeys et al. reported a glass microfluidic system in which protein digests were introduced and separated in individual channels and sequentially forwarded to a micro-scale electro-spray ionization mass spectrometer.7 Researchers have also focused efforts on transitioning multi-dimensional separations to microchips for generating high peak capacities of intact proteins required for many proteomic assays. Work in our group has generated a peak capacity of 2,660 using a polymeric microchip for the multi-dimensional separation of a fetal calf serum (FCS) sample in less than 30 min (see Figure 1).8 The microchip was fabricated in poly(methyl methacrylate) using hot embossing from a metal master.9 In this 2D format, co-migrated effluents were injected from an SDS µ-CGE 1st dimension into a micellar electrokinetic capillary chromatography 2nd dimension using SDS as the pseudo-stationary phase. As a comparison, a similar separation of the FCS sample was conducted using a conventional slab gel and IEF/SDS-PAGE. The macro-scale version produced a peak capacity of only 717 and required a ~30 h run time (see Figure 1)!

Figure 1. Left: Microchip 2D separation of the FCS proteins. 2D SDS μ-CGE x MEKC was performed at 300 V/cm and 400 V/cm, respectively. A 10 s separation time in the 1st dimension was allowed prior to performing serial 10 s MEKC cycles. A total of 159 MEKC cycles was required using a 1 s transfer time from the 1st to the 2nd dimension. The bottom panel shows a 2D map of the image displayed from the 3D landscape view (see top panel). The proteins were labeled with a fluorescent dye that covalently targeted the thiol group of cysteine amino acid residues. Right: Conventional 2D slab gel profile of the FCS proteins (bottom panel) and the corresponding 3D landscape view (top panel). The proteins here were stained with Ag for visualization.

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