Two-dimensional Separation Method Research Articles

A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification.

A two-dimensional liquid-phase separation scheme coupled with mass spectrometry (MS) is presented for proteomic analysis of cell lysates from normal and malignant breast epithelial cell lines. Liquid-phase separations consist of isoelectric focusing as the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) as the second dimension. Protein quantitation and mass measurement are performed using electrospray ionization-time of flight MS (ESI-TOF MS). Proteins are identified by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight MS (MALDI-TOF MS) and MALDI-quadrupole time of flight (QTOF)-tandem mass spectrometry (MS/MS). Two pH regions with 50-60 unique proteins in each pH range were chosen for analysis. Mass maps were created that allowed visualization of protein quantitation differences between normal and malignant breast epithelial cells. Of the approximately 110 unique proteins observed from mass mapping experiments over the limited pH range, 40 (36%) were positively identified by peptide mass fingerprinting and assigned to bands in the mass maps. Of these 40 proteins, 22 were more highly expressed in one or more of the malignant cell lines. These proteins represent potential breast cancer biomarkers that could aid in diagnosis, therapy, or drug development.

Protein pre-fractionation in detergent–polymer aqueous two-phase systems for facilitated proteomic studies of membrane proteins

Pre-fractionation of a complex mixture of proteins increases the resolution in analytical separations of proteins from cells, tissues or organisms. Here we demonstrate a novel method for pre-fractionation of membrane proteins by a detergent-based aqueous two-phase system. Membrane proteins are strongly under-represented in proteomic studies based on two-dimensional electrophoresis (2-DE). As a model system, we have isolated mitochondria from the yeast Saccharomyces cerevisiae. Mitochondrial proteins were fractionated in an aqueous two-phase system consisting of the polymer poly(ethylene glycol) and either of two commonly used non-ionic detergents, Triton X-114 or dodecyl maltoside (DDM). Soluble proteins partitioned mainly to the polymer phase while membrane proteins were enriched in the detergent phase, as identified from one-dimensional electrophoresis (1-DE) and/or 2-DE followed by mass spectrometric analysis. Pre-fractionation was further enhanced by addition of an anionic detergent, sodium dodecyl sulfate, or a chaotropic salt, NaClO 4, and by raising the pH in the system. The two-phase system pre-fractionation was furthermore combined with an alternative two-dimensional high-resolution separation method, namely ion-exchange chromatography and 1-DE. By this approach a larger number of membrane proteins could be identified compared to separation with conventional 2-DE. Thus, pre-fractionation of complex protein mixtures using the aqueous two-phase systems developed here will help to disclose larger proportions of membrane proteins in different proteomes.

Two-dimensional separation method for analysis of Bacillus subtilis metabolites via hyphenation of micro-liquid chromatography and capillary electrophoresis.

A novel two-dimensional separation method, which hyphenated chromatography and electrophoresis, was developed for analysis of Bacillus subtilis metabolites. Micro-liquid chromatography (LC) with a monolithic silica-ODS column was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Concentration strategies, namely, dynamic pH junction and sweeping, were selectively employed to interface the two dimensions, which proved to be beneficial for the detection of metabolites. For system evaluation, an artificial sample containing 54 standard metabolites was separated according to their hydrophobicity by micro-LC with gradient mode. The early-eluting fractions were separated by capillary zone electrophoresis in combination with dynamic pH junction, while the late-eluting fractions were separated by sweeping micellar electrokinetic chromatography. The middle fractions were analyzed by both modes of CE. Under the optimum conditions, all the components in the artificial sample could be well resolved. The method was applied to profile B. subtilis metabolites. Some crucial metabolites were identified. This method provided great potential for resolving complex biological samples containing compounds having different characteristics.

Two-Dimensional Chromatography as an Essential Means for Understanding Macromolecular Structure

Current liquid chromatography techniques allow to determine distributions of various properties for macromolecules. The polydisperse nature of macromolecules regulates the structure-property relationship and is responsible for the vast degree of fine-tuning of application properties. The understanding of macromolecular structure is fundamental for the use of polymers in increasingly specific applications. The coexistence of property distributions requires multi-dimensional (combined) chromatography methodologies. The use and implementation of two-dimensional (2D) separation methods and their benefits are described in this paper for synthetic polymers. Similar approaches have been used successfully for mapping complex natural and bio polymers.

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry.

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.

The Use of Sequential High-Performance Liquid Chromatography and Capillary Zone Electrophoresis to Separate the Glycosylated Peptides from Recombinant Tissue Plasminogen Activator to a Detailed Level of Microheterogeneity

The advantage of using the two-dimensional separation method was demonstrated by the separation of the complex tryptic peptides of recombinant human tissue plasminogen activator (rt-PA) glycoprotein. This method hybridizes two analytical methods where fractions containing glycopeptides are collected from reversed-phase high-performance liquid chromatography (RP-HPLC) and separated by capillary zone electrophoresis (CZE). CZE readily resolved carbohydrate structural variants, based on sialic acid content and branching, on the same peptide. Nonglycosylated peptides incidentally collected in the same RP-HPLC fraction were well resolved from the glycopeptides. This combination of RP-HPLC and CZE was able to uniquely resolve all of the peptides and glycopeptide variants in rt-PA. Confirmation of the specific peaks in the CZE was made by matrix-assisted laser-induced ionization time-of-flight mass spectrometry. The advantages and disadvantages of the existing methods for carbohydrate characterization in the biotech industry were compared. The advantage of this approach is to provide a simple extension of the existing tryptic digest protocols to include carbohydrate analysis to a detailed level of microheterogeneity.

Development of proteolytic activities during barley malting and their localization in the green malt kernel

Abstract The water-insoluble storage proteins of barley seeds reside in the starchy endosperm tissue. This tissue, when expressed from germinating barley, has a pH of 4·8. The hydrolysis of storage proteins during germination (malting) occurs mainly in the endosperm, so proteinases that are located in endosperm and are active at pH 4·8 are probably important to the storage protein hydrolytic process. This study reports our continued investigations of the endoproteinases of germinating barley (Hordeum vulgare L., cv. Morex) with a two-dimensional gel separation method that uses isoelectric focusing (IEF) and non-denaturing polyacrylamide gel electrophoresis (PAGE) in gels containing incorporated substrate protein. We identified the endoproteinases that were active at pH 4·8 and determined when they appeared during germination and where they were located in 4-day germinated barley (green malt). A total of nine cysteine, four aspartic, and two serine class proteolytic activities that were active at pH 4·8 were extracted from the endosperm tissue of green malt. It seems probable that some or all of these endosperm endoproteinases, especially proteinases C7, C8, C11, D3, E3 and E4, are the ones most intimately involved in hydrolyzing the storage proteins during malting.

Microscale structure analysis of a high-molecular-weight, hydrophobic membrane glycoprotein fraction with platelet-derived growth factor-dependent kinase activity

General methods for the study of the primary structure of picomole quantities of large, hydrophobic membrane glycoproteins with blocked amino-termini have been developed. Three techniques designed to be used in concert with each other are described: first, modified protein preparation and fragmentation techniques; secondly, a simple but very selective two-dimensional reversed-phase high-performance liquid chromatography system for the resolution of complex mixtures of small to medium-sized tryptic peptides on Vydac C 4, C 18 and diphenyl columns and thirdly, a two-dimensional separation method for large, denaturated (CNBr) polypeptide fragments by size-exclusion high-performance liquid chromatography, combined with either reversed-phase high-performance liquid chromatography (C 4) or sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with electroblotting and autoradiography. These methods were applied to studies of the platelet-derived growth factor receptor. Starting with 500 pmoles of purified protein, a total of 232 amino acids were sequenced.

The dyeing and clearing of cellulose acetate electropherograms

Automated diagonal capillary electrophoresis is a two-dimensional separation method that incorporates an immobilized enzyme reactor at the distal end of the first capillary and employs identical electrophoretic separation modes in both dimensions. Components undergo a preliminary separation in the first capillary. Fractions are parked in the reactor where some components undergo transformation. The fractions are then periodically transferred to the second capillary and replaced by the next components in the sample. Components that are not modified by the reactor will have identical mobility in both dimensions and fall on the diagonal of a reconstructed two-dimensional electropherogram, while analyte that undergoes modification will fall off the diagonal. In this study, alkaline phosphatase was immobilized in a monolithic reactor. An LTQ-Orbitrap Velos mass spectrometer was used to monitor analytes as they migrated from the second capillary. The system was used to characterize the phosphorylation status of a tryptic digest of α-casein in a background prepared from a 22-fold excess of the tryptic digest of bovine serum albumin. 120 fractions underwent automated treatment in the alkaline phosphatase reactor and separation in the second dimension capillary for over 40 min; nine phosphorylated α-casein peptides that produced 20 different phosphorylation states were detected with high confidence.