Xun Zuo

Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range two-dimensional electrophoresis.

Comprehensive analysis of complex proteomes requires prefractionation of samples prior to two‐dimensional gel electrophoresis (2‐DE). This study demonstrates the utility of using a high resolution sample prefractionation method and slightly overlapping narrow pH range two‐dimensional gel electrophoresis to enhance quantitative comparisons of complex proteomes. A key feature of this strategy is to prefractionate samples into a few well‐defined pools using microscale solution isoelectric focusing (νsol‐IEF) prior to 2‐DE protein analysis. Sample prefractionation is achieved using a series of tandem small volume chambers (500 νL) separated by thin membranes containing immobilines at specific pH’s. The resulting well‐resolved fractionated samples are optimally separated on a series of slightly overlapping narrow pH range immobilized pH gradient (IPG) gels, which are approximately 0.1 pH units wider than the νsol‐IEF fractionated pools. When νsol‐IEF prefractionation was applied to proteome analyses of mouse serum, it resulted in the capacity to separate much higher protein loads on narrow pH range IPG gels while retaining good resolution and spot recovery. More importantly, the prefractionation of serum greatly enhanced the ability to detect low abundance proteins, because major interfering proteins were removed from most fractions. At least 6‐ to 30‐fold higher protein loads were possible for nonalbumin fractions on narrow pH range IPG gels. The dynamic range of protein detection is substantially increased since higher protein loads can be applied to narrow pH range 2‐DE gels, and duplicate gels can be stained with colloidal Coomassie and silver stains for quantitation of abundant and minor proteins, respectively. Finally, the ability to effectively fractionate complex proteomes into very narrow ranges (< 0.5 pH units) strongly suggests that νsol‐IEF could be used to prefractionate complex samples for subsequent direct analysis by liquid chromatography‐tandem mass spectrometry methods as an alternative to using overlapping narrow pH range 2‐DE gels.

Quantitative evaluation of protein recoveries in two-dimensional electrophoresis with immobilized pH gradients.

In this study, metabolically radiolabeled Escherichia coli cell extracts were used to systematically evaluate protein recoveries at each step of two‐dimensional (2‐D) electrophoresis and using different sample application methods. Sample application using sample cups resulted in better protein recovery compared with sample loading by rehydration when the Multiphor system was used. At least 50% or more of an E. coli extract was lost when high protein amounts (500 μg) were loaded by rehydration using this system, which employs separate holders for rehydration and isoelectric focusing (IEF). In contrast, when the IPGphor system was used, rehydration sample loading consistently yielded the highest overall protein recoveries. These improved protein recoveries were due to integration of rehydration and electrophoretic separation in a single unit. Even at high protein loads (500 μg), less than 15—20% of the proteins were lost when proteins were loaded by rehydration using sample buffer containing 2% carrier ampholytes in the ceramic immobilized pH gradient (IPG) strip holders used for both rehydration and IEF. Regardless of the loading conditions used, carrier ampholytes in the sample buffer increased protein recoveries. Use of thiourea did not significantly affect protein recoveries but did improve protein resolution in 2‐D gels as expected. In summary, these results show the best protein recoveries are obtained for all protein loads when samples are applied to IPG strips during rehydration using a single device for both rehydration and IEF. In contrast, the poorest recoveries are obtained when rehydration and IEF are performed in separate devices, and losses increase dramatically with increasing protein loads using this approach.

Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels.

Current methods for quantitatively comparing proteomes (protein profiling) have inadequate resolution and dynamic range for complex proteomes such as those from mammalian cells or tissues. More extensive profiling of complex proteomes would be obtained if the proteomes could be reproducibly divided into a moderate number of well-separated pools. But the utility of any prefractionation is dependent upon the resolution obtained because extensive cross contamination of many proteins among different pools would make quantitative comparisons impractical. The current study used a recently developed microscale solution isoelectrofocusing (musol-IEF) method to separate human breast cancer cell extracts into seven well-resolved pools. High resolution fractionation could be achieved in a series of small volume tandem chambers separated by thin acrylamide partitions containing covalently bound immobilines that establish discrete pH zones to separate proteins based upon their pIs. In contrast to analytical 2-D gels, this prefractionation method was capable of separating very large proteins (up to about 500 kDa) that could be subsequently profiled and quantitated using large-pore 1-D SDS gels. The pH 4.5-6.5 region was divided into four 0.5 pH unit ranges because this region had the greatest number of proteins. By using very narrow pH range fractions, sample amounts applied to narrow pH range 2-D gels could be increased to detect lower abundance proteins. Although 1.0 pH range 2-D gels were used in these experiments, further protein resolution should be feasible by using 2-D gels with pH ranges that are only slightly wider than the pH ranges of the musol-IEF fractions. By combining musol-IEF prefractionation with subsequent large pore 1-D SDS-PAGE (>100 kDa) and narrow range 2-D gels (<100 kDa), large proteins can be reliably quantitated, many more proteins can be resolved, and lower abundance proteins can be detected.

Overview of proteome analysis.

This unit reviews the new discipline of proteomics, which includes any large‐scale protein‐based systematic analysis of the proteome or defined sub‐proteome from a cell, tissue, or entire organism. Proteomics originated in the mid‐1990s due to two key enabling advances, availability of complete genome sequences, and mass spectrometry advances that allowed high sensitivity identifications of proteins. Proteome analyses can be broadly categorized into three types of studies: quantitative protein profile comparisons, analysis of protein‐protein interactions, and compositional analysis of simple proteomes or subproteomes such as organelles or large protein complexes. The complexity of different types of proteomes, the merits of targeted versus global proteome studies, and the advantages of alternative separation and analysis technologies are discussed.

Electrophoretic Prefractionation for Comprehensive Analysis of Proteomes

Publisher Summary Two-dimensional polyacrylamide gel electrophoresis (2DE) has dominated protein profile analysis for more than 25 years, and it is still the method of choice in many laboratories for quantitatively comparing changes of proteins for proteome analysis experiments. Due to the limited capacities of both gel based and nongel based protein profiling methods, it has become apparent that more powerful and reliable methods are needed for prefractionation of complex proteomes prior to 2D gels and alternative LC–MS analysis. Although preparative IEF prefractionation methods are not orthogonal to 2D gels, they show the most promise due to the very high resolution that can be obtained. Alternative lower resolution prefractionation methods severely compromise the ability to perform comprehensive quantitative comparisons due to variable cross-contamination between adjacent fractions and greater fraction complexity. A number of preparative solution-based IEF methods have been productively integrated into quantitative protein profiling strategies including Rotofor, FFE, IsoPrime, the MCE and μsol-IEF. However, some of these methods require large sample amounts and result in large dilute fractions that are not compatible with direct analysis using downstream protein profiling methods.

Protein Profiling by Microscale Solution Isoelectrofocusing (MicroSol‐IEF)

Sample prefractionation is essential for more comprehensive coverage and reliable detection of low‐abundance proteins in complex proteomes. An efficient and reproducible new method for sample prefractionation is microscale solution isoelectrofocusing (MicroSol‐IEF), in which samples are separated into chambers defined by membranes of specific pH, yielding well resolved fractions on the basis of isoelectric point (pI). The output seamlessly interfaces with narrow‐pH‐range 2‐D gels, enhancing data obtained from protein profiling studies, including quantitative proteome comparisons. This unit presents the MicroSol‐IEF method using the ZOOM IEF Fractionator with either commercially available or custom‐made pH partition membranes. Alternative configurations are possible for separating samples into different numbers of fractions with various pH ranges and volumes. A detailed method is provided for preparing custom pH membranes. In addition, methods are provided for evaluating the effectiveness of the prefractionation, using 1‐D and 2‐D gel electrophoresis. Approaches for quantitative protein profiling that incorporate MicroSol‐IEF are also discussed.