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.

Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma.

Systematic detection of low-abundance proteins in human blood that may be putative disease biomarkers is complicated by an extremely wide range of protein abundances. Hence, depletion of major proteins is one potential strategy for enhancing detection sensitivity in serum or plasma. This study compared a recently commercialized HPLC column containing antibodies to six of the most abundant blood proteins ("Top-6 depletion") with either older Cibacron blue/Protein A or G depletion methods or no depletion. In addition, a prototype spin column version of the HPLC column and an alternative prototype two antibody spin column were evaluated. The HPLC polyclonal antibody column and its spin column version are very promising methods for substantially simplifying human serum or plasma samples. These columns show the lowest nonspecific binding of the depletion methods tested. In contrast other affinity methods, particularly dye-based resins, yielded many proteins in the bound fractions in addition to the targeted proteins. Depletion of six abundant proteins removed about 85% of the total protein from human serum or plasma, and this enabled 10- to 20-fold higher amounts of depleted serum or plasma samples to be applied to 2-D gels or alternative protein profiling methods such as protein array pixelation. However, the number of new spots detected on 2-D gels was modest, and most newly visualized spots were minor forms of relatively abundant proteins. The inability to detect low-abundance proteins near expected 2-D staining limits was probably due to both the highly heterogeneous nature of most plasma or serum proteins and masking of many low-abundance proteins by the next series of most abundant proteins. Hence, non2-D methods such as protein array pixelation are more promising strategies for detecting lower abundance proteins after depleting the six abundant proteins.

A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes.

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO-approved criteria for high-confidence protein assignments. Most importantly, a substantial number of low-abundance proteins (low ng/mL - pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 microL (45 microg) of plasma and 2.4 microL (204 microg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.

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-1990 s 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.

Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation.

The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409-426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the "marked box" region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2cyclin DE. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.