PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 16737203

Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips.

Hamed Shadpour·Steven A Soper
View original source ↗Export metadata ↓

Abstract

The work presented herein describes highly efficient, two-dimensional (2D) electrophoretic separations of proteins in a PMMA-based microchip. Sodium dodecyl sulfate microcapillary gel electrophoresis (SDS micro-CGE) and micellar electrokinetic chromatography (MEKC) were used as the separation modes for the first and second dimension of the electrophoresis, respectively. The microchip was prepared by hot embossing into PMMA from a brass mold master fabricated via high-precision micromilling. The microchip incorporated a 30-mm SDS micro-CGE and a 10-mm MEKC dimension length. Electrokinetic injection and separation were used with field strengths of up to 400 V/cm. Alexa Fluor 633 conjugated proteins, ranging in size from 38 to 110 kDa, were detected using laser-induced fluorescence with excitation/emission at 633/652 nm. Average plate numbers (N) of 4.8 x 10(4) and 1.2 x 10(4) were obtained in the SDS micro-CGE and MEKC separation dimensions, respectively, for the investigated proteins corresponding to plate heights (H) of 0.62 and 0.87 microm. Effluents from the first dimension (SDS micro-CGE) were repetitively transferred into the second dimension every 0.5 s of run time in the first dimension with the electrophoresis run time in the MEKC dimension being 10 s. The 2D separation was performed on the investigated proteins in approximately 12 min and provided a peak capacity of approximately 1000.

Explore related subjects

Electrophoresis, Gel, Two-DimensionalPolymethyl MethacrylateProtein Array AnalysisProteinsJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Hamed Shadpour, Steven A Soper. 2006-06-01. Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips.. https://doi.org/10.1021/ac0600398

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Proteomic response to intracellular proteins of Monascus pilosus grown under phosphate-limited complex medium with different growth rates and pigment production.

Monascus pigments are important colorings in food applications. Rice containing potassium phosphate and sodium nitrate was reported as a good pigment-producing medium for Monascus in previous studies. We found that the lack of potassium phosphate in this medium depressed red pigment production in cultivated Monascus pilosus. However, the influence of phosphate limitation on the biochemical metabolisms concerning culture growth and pigment production in Monascus remains unknown. Here, we used proteomic analysis by two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), tandem mass spectrometry (MS/MS), and database interrogation to separate and identify the proteins of M. pilosus grown between the lack of potassium phosphate and the control media. Phosphate limitation to this complex medium induced an up-regulation of aldehyde dehydrogenase and several glycolytic enzymes in Monascus relative to the control. In contrast, the metabolic enzymes such as glucosamine:fructose-6-phosphate aminotransferase and ADP-ribosylation factor 1 were up-regulated in the control.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2007

Toward an integrated microchip sized 2-D polyacrylamide slab gel electrophoresis device for proteomic analysis.

We describe a miniaturized instrument capable of performing 2-DE. Our miniaturized device is able to perform IEF and polyacrylamide slab gel electrophoresis (PASGE) in the same unit. It consists of a compartment for a first-dimensional IEF gel, which is connected to a second-dimensional PASGE gel. The focused samples are automatically transferred from the IEF gel to the PASGE gel by electromigration. Our preliminary experiments show that the device is able to focus and separate a mixture of proteins in approximately 1 h, excluding the time required for the staining procedure. On average, the gel-to-gel retardation factor (Rf) variation was 6.2% (+/-0.9%) and pI variation was 2.5% (+/-0.6%). Separated protein spots were excised from stained gels, digested with trypsin, and further identified by MS, thus enabling direct proteomic analysis of the separated proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2007

Proteomic analysis of human very low-density lipoprotein by two-dimensional gel electrophoresis and MALDI-TOF/TOF.

Biochemical studies of lipoproteins have shed light on their composition, highly contributing to the comprehension of their function. Due to the complexity of their structure, however, an in-depth structural analysis, in terms of components and PTMs, may still unravel important players in physiological and pathological processes of lipid metabolism. In this study, we performed a protein map of very low-density lipoprotein (VLDL) using a 2-DE MALDI-TOF/TOF proteomic approach. Several VLDL-associated apolipoproteins were identified, including five isoforms of apoE, three isoforms of apoC-IV, and one isoform each of apoC-III, apoM, apoA-I, and apoA-IV. Notably, we also identified seven isoforms of apoL-I and two isoforms of prenylcysteine lyase as new VLDL-associated proteins. Furthermore, we were able to identify PTM of apoE, which was found to be differently O-glycosylated at Thr212 residue, and PTM of apoL-I which we described, for the first time, to be phosphorylated at Ser296. While the physiological relevance of our finding remains to be assessed, we believe that our results will be useful as reference for future studies of VLDL structure in specific physiopathological conditions.

Electrophoresis, Gel, Two-Dimensional↗