Free-flow electrophoresis in the proteomic era: a technique in flux.

Since its introduction five decades ago, free-flow electrophoresis (FFE) has been mainly employed for the isolation and fractionation of cells, cell organelles and protein mixtures. In the meantime, the growing interest in the proteome of these bio-particles and biopolymers has shed light on two further facets in the potential of FFE, namely its applicability as an analytical tool and sensor. This review is intended to outline recent innovations, FFE has gained in the proteomic era, and to point out the valuable contributions it has made to the analysis of the proteome of cells, sub-cellular organelles and functional protein networks.

Peroxisomes from the heavy mitochondrial fraction: isolation by zonal free flow electrophoresis and quantitative mass spectrometrical characterization.

Peroxisomes are a heterogeneous group of organelles fulfilling reactions in a variety of metabolic pathways. To investigate if functionally different subpopulations can be found within a single tissue, peroxisomes from the heavy mitochondrial fraction (HM-Po) of the rat liver were isolated and compared to "classic" peroxisomes from the light mitochondrial fraction (LM-Po) using iTRAQ tandem mass spectrometry. Peroxisomes represent only a minor although significant proportion of the heavy mitochondrial fraction (2700g(max)) precluding a straightforward isolation by standard protocols.

Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS.

Early diagnosis of life-threatening invasive aspergillosis in neutropenic patients remains challenging because current laboratory methods have limited diagnostic sensitivity and/or specificity. Aspergillus species are known to secrete various pathogenetically relevant proteases and the monitoring of their protease activity in serum specimens might serve as a new diagnostic approach.For the characterization and identification of secreted proteases, the culture supernatant of Aspergillus fumigatus was fractionated using free flow electrophoresis (Becton Dickinson).

Functional and complementary phosphorylation state attributes of human insulin-like growth factor-binding protein-1 (IGFBP-1) isoforms resolved by free flow electrophoresis.

Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size. High concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) have been associated with FGR. Phosphorylation of IGFBP-1 is a mechanism by which insulin-like growth factor-I (IGF-I) bioavailability can be modulated in FGR.

Isolation of organelles and prefractionation of protein extracts using free-flow electrophoresis.

One of the major obstacles in the analysis of proteomes is the extreme complexity of any particular cell or biological fluid. Free-flow electrophoresis (FFE) is a powerful tool for reduction of this complexity, which is a prerequisite for systematic and comprehensive protein analyses. Protocols are provided in this unit for sample fractionation at two different stages: on the protein level by isoelectric focusing FFE fractionation of crude protein mixtures such as whole cell lysates, and on a subcellular level by zone-electrophoretic FFE purification of organelles.

Free-flow electrophoresis system for plasma proteomic applications.

This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate human plasma for proteome analysis. FFE is a highly versatile technology applied in the field of proteomics because of its continuous processing of sample and high resolution in separation of most kinds of charged or chargeable particles including ions, proteins peptides, organelles, and whole cells. FFE is carried out in an aqueous medium without inducing any solid matrix, such as acrylamide, so that it simplifies complex sample for the downstream analysis.

Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis.

Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins.

Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis.

Multidimensional LC-MS based shotgun proteomics experiments at the peptide level have traditionally been carried out by ion exchange in the first dimension and reversed-phase liquid chromatography in the second. Recently, it has been shown that isoelectric focusing (IEF) is an interesting alternative approach to ion exchange separation of peptides in the first dimension. Here we present an improved protocol for peptide separation by continuous free-flow electrophoresis (FFE) as the first dimension in a two-dimensional peptide separation work flow.

Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3).

The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step.

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing.

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient.

In-gel digestion of proteins using a solid-phase extraction microplate.

We present a new procedure for in-gel digestion of proteins introducing a combination of two different 96-well microplates. The two plates have incorporated small capillaries with a length of 2.4 mm in each well, one of which has 75-microm-inner diameter capillaries, whereas the second plate has reversed-phase-type capillaries fixed to it.

Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis.

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity.

Proteome analysis of the thermoreceptive pit membrane of the western diamondback rattlesnake Crotalus atrox.

Rattlesnakes detect their prey's temperature by means of a cavern-like structure, the pit organ. The sensory component of this organ lies within a thin membrane called the pit membrane. Proteome analysis conducted on this neurosensory tissue revealed only a relatively small number of proteins, thereby depicting its high degree of specialization.

A microsomal ecdysone-binding cytochrome P450 from the insect Locusta migratoria purified by sequential use of type-II and type-I ligands.

A dual-affinity method was established to purify, for the first time, a microsomal ecdysone-binding cytochrome P450 protein from locust Malpighian tubules. This method involved, after prepurification on omega-octylamino-agarose and hydroxylapatite, binding of cytochrome P450 to an immobilized triazole-based general P450 inhibitor (type-II ligand) followed by elution with the substrate ecdysone (type-I ligand) of the bound cytochrome. The isolated material showed a typical cytochrome P450 spectrum, a specific heme content of 13 nmol/mg protein, and a prominent protein of about 60 kDa on SDS-PAGE.

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library.

Human CLP36, a PDZ-domain and LIM-domain protein, binds to alpha-actinin-1 and associates with actin filaments and stress fibers in activated platelets and endothelial cells.

A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36. The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells.

Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins.

The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N.

Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily.

Identification of in vivo substrates of the chaperonin GroEL.

The chaperonin GroEL has an essential role in mediating protein folding in the cytosol of Escherichia coli. Here we show that GroEL interacts strongly with a well-defined set of approximately 300 newly translated polypeptides, including essential components of the transcription/translation machinery and metabolic enzymes. About one third of these proteins are structurally unstable and repeatedly return to GroEL for conformational maintenance.

Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism?

Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.

Alkaline density gradient floatation of membranes: polypeptide composition of the mammalian peroxisomal membrane.

A method for purification of the peroxisomal membrane from rat liver is described. The procedure consists of floating the (contaminated) peroxisomal membranes through an alkaline sucrose density gradient. A good resolution between the peroxisomal membrane and other membrane systems is achieved.

Subunit C of the vacuolar H+-ATPase of Hordeum vulgare.

The molecular cloning of the first subunit C of the plant vacuolar H+-ATPase is reported. Tonoplast vesicles were purified from barley leaves by sucrose gradient centrifugation, and the tonoplast polypeptides were separated by two-dimensional (2-D) gel electrophoresis. Using an anti-ATPase holoenzyme antibody, a polypeptide was recognized in the molecular mass range of 40 kDa with an isoelectric point of about 6.

Identification of a 24 kDa intrinsic membrane protein from mammalian peroxisomes.

A 24 kDa protein from rat liver peroxisomal membrane was isolated and subjected to Edman degradation. Using the N-terminal sequence of this polypeptide we have identified several rat and human expressed sequence tags in the GenBank Database. The complete sequence of a human cDNA clone was determined.