In-gel peptide IEF sample preparation for LC/MS analysis.

The technique of proteolytically digesting a sample and identifying its protein components by liquid chromatography followed by mass spectrometry (LC-MS) is a widely used analytical tool. Prior fractionation by isoelectric focusing (IEF) may be performed to increase the depth of proteome coverage. Here, we describe a method for in-gel IEF separation of a proteolytic digest that utilizes commercially available immobilized pH gradient (IPG) strips and a widely used IEF instrument.

The chromosome-centric human proteome project: a call to action.

The grand vision of the human proteome project (HPP) is moving closer to reality with the recent announcement by HUPO of the creation of the HPP consortium in charge of the development of a two-part HPP, one focused on the description of proteomes of biological samples or related to diseases (B/D-HPP) and the other dedicated to a systematic description of proteins as gene products encoded in the human genome (the C-HPP). This new initiative of HUPO seeks to identify and characterize at least one representative protein from every gene, create a protein distribution atlas and a protein pathway or network map. This vision for proteomics can be the roadmap of biological and clinical research for years to come if it delivers on its promises.

Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT.

Ideally, shotgun proteomics would facilitate the identification of an entire proteome with 100% protein sequence coverage. In reality, the large dynamic range and complexity of cellular proteomes results in oversampling of abundant proteins, while peptides from low abundance proteins are undersampled or remain undetected. We tested the proteome equalization technology, ProteoMiner, in conjunction with Multidimensional Protein Identification Technology (MudPIT) to determine how the equalization of protein dynamic range could improve shotgun proteomics methods for the analysis of cellular proteomes.

MicroPrep: chip-based dielectrophoretic purification of mitochondria.

We have developed a microfluidic system--microPrep--for subcellular fractionation of cell homogenates based on dielectrophoretic sorting. Separation of mitochondria isolated from a human lymphoblastoid cell line was monitored by fluorescence microscopy and further characterized by western blot analysis. Robust high throughput and continuous long-term operation for up to 60 h of the microPrep chip system with complex biological samples became feasible as a result of a comprehensive set of technical measures: (i) coating of the inner surfaces of the chip with BSA, (ii) application of mechanical actuators to induce periodic flow patterns, (iii) efficient cooling of the device to ensure integrity of organelle, (iv) a wide channel to provide for high fluidic throughput, and (v) integration of a serial arrangement of 10 dielectrophoretic deflector units to enable separation of samples with a high particle load without clogging.

Enriching basic and acidic rat brain proteins with ion exchange mini spin columns before two-dimensional gel electrophoresis.

Proteome analysis by two-dimensional gel electrophoresis (2-DGE) faces significant challenges because of the complexity of biological samples. However, the complexity of a protein sample can be reduced prior to 2-DGE by applying protein fractionation. Protein fractionation allows analysis of one protein subset at a time, thereby, increasing the load of proteins of interest, enriching low-abundance proteins, and increasing the resolution of protein spots on a 2-D gel.

Increase in local protein concentration by field-inversion gel electrophoresis.

Background: Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).

Actin deficiency induces cofilin phosphorylation: proteome analysis of HeLa cells after beta-actin gene silencing.

Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis.