Hollow-fiber flow/hyperlayer field-flow fractionation for the size characterization of airborne particle fractions obtained by SPLITT fractionation

Hollow-fiber flow field-flow fractionation (HF FlFFF) was applied for the separation and size characterization of airborne particles which were collected in a municipal area and prefractionated into four different-diameter intervals >5.0, 2.5-5.0, 1.5-2.5, <1.5 microm) by continuous split-flow thin (SPLIIT) fractionation. Experiments demonstrated the possibility of utilizing a hollow-fiber module for the high-performance separation of supramicron-sized airborne particles at steric/hyperlayer operating mode of HF FlFFF. Eluting particles during HF FlFFF separation were collected at short time intervals (approximately 10 s) for the microscopic examination. It showed that particle size and size distributions of all SPLITT fractions of airborne particles can be readily obtained using a calibration and that HF FlFFF can be utilized for the size confirmation of the sorted particle fraction during SPLITT fractionation.     

Flow field-flow fractionation for the analysis and characterization of natural colloids and manufactured nanoparticles in environmental systems: a critical review

The use of flow field flow fractionation (FlFFF) for the separation and characterization of natural colloids and nanoparticles has increased in the last few decades. More recently, it has become a popular method for the characterization of manufactured nanoparticles. Unlike conventional filtration methods, FlFFF provides a continuous and high-resolution separation of nanoparticles as a function of their diffusion coefficient, hence the interest for use in determining particle size distribution. Moreover, when coupled to other detectors such as inductively coupled plasma-mass spectroscopy, light scattering, UV-absorbance, fluorescence, transmission electron microscopy, and atomic force microscopy, FlFFF provides a wealth of information on particle properties including, size, shape, structural parameters, chemical composition and particle-contaminant association. This paper will critically review the application of FlFFF for the characterization of natural colloids and natural and manufactured nanoparticles. Emphasis will be given to the detection systems that can be used to characterize the nanoparticles eluted from the FlFFF system, the obtained information and advantages and limitation of FlFFF compared to other fractionation and particle sizing techniques. This review will help users understand (i) the theoretical principles and experimental consideration of the FlFFF, (ii) the range of analytical tools that can be used to further characterize the nanoparticles after fractionation by FlFFF, (iii) how FlFFF results are compared to other analytical techniques and (iv) the range of applications of FlFFF for natural and manufactured NPs.     

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.     

New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues

To clarify the mechanism of the anticarcinogenic effect of conjugated linoleic acid (CLA), its intracellular distribution needs to be determined. Subcellular fractionation using centrifugation techniques is a method that is frequently used for isolation of cell organelles from different tissues. But as the size and density of the organelles differ, the method needs to be optimised for every type of tissue. The novelty of this study is the application of a subcellular fractionation method to human healthy and cancerous renal and testicular tissue. Separation of total tissue homogenate into nuclei, cytosol, and a mixture of mitochondria and plasma membranes was achieved by differential centrifugation. As mitochondria and plasma membranes seemed to be too similar in size and weight to be separated by differential centrifugation, discontinuous density-gradient centrifugation was carried out successfully. The purity of the subcellular fractions was checked by measuring the activity of marker enzymes. All fractions were highly enriched in their corresponding marker enzyme. However, the nuclear fractions of kidney and renal cell carcinoma were slightly contaminated with mitochondria and plasma membrane fractions of all tissues with lysosomes. The fraction designated the cytosolic fraction contained not only cytosol, but also microsomes and lysosomes. The CLA contents of the subcellular fractions were in the range 0.13–0.37% of total fatty acids and were lowest in the plasma membrane fractions of all types of tissue studied. C16:0, C18:0, C18:1 c9, C18:2 n-6, and C20:4 n-6 were found to be the major fatty acids in all the subcellular fractions studied. However, marked variations in fatty acid content between subcellular fractions and between types of tissue were detectable. Because of these differences between tissues, no general statement on characteristic fatty acid profiles of single subcellular fractions is possible.     

Effect of sodium dodecyl sulfate on protein separation by hollow fiber flow field-flow fractionation

Effects of protein denaturation and formation of protein-sodium dodecyl sulfate (SDS) complexes on protein separation and identification were investigated using hollow fiber flow field-flow fractionation (HF5) and nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). Denaturation and formation of protein-SDS complexes prior to HF5 separation resulted an increase in the retention of few protein standards due to unfolding of the protein structures and complexation, yielding ~30% increase in hydrodynamic diameter. In addition, low molecular weight proteins which could be lost from the HF membrane due to the pore size limitation showed an increase of peak recovery about 2-6 folds for cytochrome C and carbonic anhydrase. In the case of proteins composed of a number of subunits, denaturation resulted in a decrease in retention due to dissociation of protein subunits. A serum proteome sample, denatured with dithiothreitol and SDS, was fractionated by HF5, and the eluting protein fractions after tryptic digestion were analyzed for protein identification using nLC-ESI-MS-MS. The resulting pools of identified proteins were found to depend on whether the serum sample was treated with or without denaturation prior to the HF5 run due to differences in the aqueous solubility of the proteins. The enhancement of protein solubility by SDS also increased the number of identified membrane proteins (54 vs. 31).     

Speciation and subcellular location of Se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic fluorescence spectrometric detection

Speciation of Se-containing proteins in the subcellular fractions of human liver was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by hydride generation-atomic fluorescence spectrometric (HG-AFS) detection. It was found that about 24 kinds of Se-containing proteins existed in subcellular fractions of normal human liver. The molecular weights (MW) of the subunits were mostly in the range 20-30 kDa and 50-80 kDa. Major Se-containing protein fractions at 61 kDa and 21 kDa are probably selenoprotein P and glutathione peroxidase, respectively. The 54 kDa protein is probably a thioredoxin reductase, which is presented in nuclei, mitochondria, lysosome, microsome and cytosol. We noticed that the Se-containing protein with the lowest MW of 9.3 kDa only existed in lysosome. Most of the proteins have not been identified and would require further investigation to characterize them. The specific subcellular distributions of different Se-containing proteins suggest that they could play important biological roles in each organelle.     

Natural sample fractionation by FlFFF-MALLS-TEM: sample stabilization, preparation, pre-concentration and fractionation

Two flow field flow fractionation (FlFFF) systems: symmetrical (SFlFFF) and asymmetrical (ASFlFFF) were evaluated to fractionate river colloids. Samples stability during storage and colloids concentration are the main challenges limiting their fractionation and characterization by FlFFF. A pre-fractionation (<0.45 microm) and addition of a bactericide such as NaN3 into river colloidal samples allowed obtaining stable samples without inducing any modification to their size. Stirred cell ultra-filtration allowed colloidal concentration enrichment of 25-folds. Scanning electron microscope (SEM) micrographs confirmed the gentle pre-concentration of river samples using the ultra-filtration stirred cell. Additionally, larger sample injection volume in the case of SFlFFF and on channel concentration in the case of ASFlFFF were applied to minimize the required pre-concentration. Multi angle laser light scattering (MALLS), and transmission electron microscope (TEM) techniques are used to evaluate FlFFF fractionation behavior and the possible artifacts during fractionation process. This study demonstrates that, FlFFF-MALLS-TEM coupling is a valuable method to fractionate and characterize colloids. Results prove an ideal fractionation behavior in case of Brugeilles sample and steric effect influencing the elution mode in case of Cézerat and Chatillon. Furthermore, comparison of SFlFFF and ASFlFFF fractograms for the same sample shows small differences in particle size distributions.     

Off-line and on-line assay of membrane protein with o-phthaldialdehyde by flow-injection with post-column reaction

A rapid and convenient flow-injection absorption spectrosphotometric procedure was developed for the determination of protein and peptide concentrations in discrete samples that contain biological membranes (subcellular particles) which scatter light. With some modification, the same system serves as a post-column reactor to determine protein and peptide concentrations continuously in the effluent of a separation device. The procedure is based on the well known determination of primary amines by reaction with a thiol and o-phthaldialdehyde. Although designed for preparations of biological membranes, the procedure is applicable to any solution in which the primary amino groups are predominantly those belonging to the proteins and/or peptides present; this includes many protein solutions which are turbid at acidic or neutral pH but clear at alkaline pH. The off-line flow-injection procedure for discrete samples and the on-line post-column reactor for flowing column effluents have been found especially useful for determining the distribution of membrane protein in the chanel effluent after fractionation of subcellular particle preparations containing corn root mitochondria and microsomes.     

Characterization of functionalized styrene-butadiene rubber by flow field-flow fractionation/light scattering in organic solvent

Flow field-flow fractionation (FlFFF) using an organic solvent as mobile phase has been effectively utilized for the separation and characterization of functionalized styrene-butadiene rubbers (SBR) that are polymerized and followed by coupling reaction in solution. Separation of broad molecular weight SBR was accomplished by an asymmetrical FlFFF channel in THF under field programming and the molecular weight distribution (MWD) of the SBR sample was determined by on-line measurement of light scattering. In this study, FlFFF has been utilized to characterize high-MW functionalized SBR from the low-MW non-functionalized molecules which were used for coupling reaction to produce high-MW functionalized SBRs, and to determine the coupling number of the functionalized SBRs depending on the type of the coupling reagents. The resulting MWD of the SBR samples prepared by the different coupling reagents (SnCl(4) and a polydimethylsiloxane compound) were compared.     

Application of flow field-flow fractionation for the characterization of macromolecules of biological interest: a review

An overview is given of the recent literature on (bio) analytical applications of flow field-flow fractionation (FlFFF). FlFFF is a liquid-phase separation technique that can separate macromolecules and particles according to size. The technique is increasingly used on a routine basis in a variety of application fields. In food analysis, FlFFF is applied to determine the molecular size distribution of starches and modified celluloses, or to study protein aggregation during food processing. In industrial analysis, it is applied for the characterization of polysaccharides that are used as thickeners and dispersing agents. In pharmaceutical and biomedical laboratories, FlFFF is used to monitor the refolding of recombinant proteins, to detect aggregates of antibodies, or to determine the size distribution of drug carrier particles. In environmental studies, FlFFF is used to characterize natural colloids in water streams, and especially to study trace metal distributions over colloidal particles. In this review, first a short discussion of the state of the art in instrumentation is given. Developments in the coupling of FlFFF to various detection modes are then highlighted. Finally, application studies are discussed and ordered according to the type of (bio) macromolecules or bioparticles that are fractionated.     

结晶分级技术在支化聚乙烯研究中的应用

介绍了近年发展起来的几种结晶分级技术及其在支化聚乙烯结构表征及性能研究方面的应用。利用升温淋洗分级技术(TREF),可根据结晶特性的不同将高分子分离成多个分布较窄的级份,通过分别表征各级份的链结构,从而可获得高分子链结构方面较为准确的信息。基于差示扫描量热技术(DSC)发展起来的两类热分级技术,主要包括逐步结晶热分级(SC)和连续自成核退火分级(SSA)技术,虽然不能从物理上对高分子进行分级,但通过选择适当的操作参数,也能得到一系列与升温淋洗分级实验类似的链结构信息,并且具有设备简单、操作方便、样品用量少、耗时短等优点。本文结合我们自己的工作,对各种分级技术的原理、实验操作及应用进行了系统综述,并展望了结晶分级技术发展的某些可能趋势。     

Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.     

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e. 1D SDS-PAGE, 1D preparative SDS-PAGE, IEF-IPG, and 2D-PAGE in their performance as fractionation approaches in nano LC-ESI-MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS-PAGE and IEF-IPG had the highest number of identifications. The IEF-IPG technique resulted in the highest average number of detected peptides per protein. The 2D-PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS-PAGE and IEF-IPG for the improved sensitivity of profiling without significant decrease in throughput.     

Hen egg white fractionation by ion-exchange chromatography

Major hen egg white proteins have been widely studied for their functional properties but these studies still are unable to explain, alone, all of the biological properties of hen egg white. Hence, it is still interesting to produce pure and non-altered proteins to improve our knowledge on the biological properties of hen egg white. Presently, identification and characterization of both bioactive peptides and minor proteins from hen egg white is essential work for progressing in the understanding of hen egg white biological properties. With this objective in mind, a new process for a complete "mucin free" hen egg white fractionation based on ion exchange chromatography is proposed. "Mucin free" egg white is fractionated into six different fractions. Four of them are high-recovery yield purified fractions of lysozyme, ovotransferrin, ovalbumin and flavoprotein. The two other fractions are enriched in recently detected minor proteins in hen egg white.     

Differential Proteins of the Optic Ganglion in Octopus vulgaris Under Methanol Stress Revealed Using Proteomics

An analytical approach using the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique separated the proteome from the optic ganglia of Octopus vulgaris (OVOG). Approximately 600 protein spots were detected from the extraction when applying 150 μg protein to a 2D-PAGE gel in the pH range 5.0-8.0. Compared to the control, significant changes of 18 protein spots were observed in OVOG under the stress of native seawater containing 2% methanol for 72 h. Among these spots, we found that eight were down-regulated and ten were up-regulated in the gels, which were further identified using both peptide mass fingerprinting and database searches. Significant proteins such as glyceraldehyde-3-phosphate dehydrogenase, alpha subunit of succinyl-CoA synthetase, alcohol dehydrogenase, and long-chain specific acyl-CoA dehydrogenase were up-regulated proteins, whereas putative ABC transporter was a down -regulated protein. These differential proteins at the level of subcellular localization were further classified using LOCtree software with a hierarchical system of support vector machines. We found that most of the differential proteins in the gel could be identified as mitochondrial proteins, suggesting that these protective or marker proteins might help to prevent methanol poisoning via the mitochondria in the optical ganglia. The results indicated that both beta-tubulin and beta-actin were potential biomarkers as up-regulated proteins for monitoring methanol toxicosis associated with fish foods such as octopus and shark.     

Flow field-flow fractionation: a versatile approach for size characterization of α-tocopherol-induced enlargement of gold nanoparticles

Flow field-flow fractionation (FlFFF) was used for size characterization of gold nanoparticles. The measured particle sizes obtained from FlFFF for the commercial 10 nm gold nanoparticle standard and the gold nanoparticles synthesized in the laboratory were in good agreement with those measured by transmission electron microscopy (TEM). Further, the capability of α-tocopherol to induce enlargement of gold nanoparticles by catalysis of the reduction of AuCl₄⁻ by citrate was observed by monitoring the changes in particle size of gold nanoparticles using FlFFF. The effects of α-tocopherol and incubation time on enlargement of the gold nanoparticles were examined. Higher concentrations of α-tocopherol resulted in larger nanoparticles. At fixed α-tocopherol concentration, larger nanoparticles were formed at longer incubation times.     

Flow field-flow fractionation with off-line electrothermal atomic absorption spectrometry for size characterization of silver nanoparticles

Flow field-flow fractionation (Fl-FFF) with off-line electrothermal atomic absorption spectrometry (ETAAS) detection was developed and employed for particle size characterization of Ag NPs stabilized by citrate, pectin, and alginate. Citrate stabilized-Ag NPs were prepared from sodium borohydride reduction of silver nitrate. Sodium citrate was used as the capping agent to stabilize Ag NPs and prevent the aggregation process. Pectin stabilized- and alginate stabilized-Ag NPs were prepared from ascorbic acid reduction of silver nitrate. Pectin and alginate were used as the capping agent for pectin stabilized- and alginate stabilized-Ag NPs, respectively. Three types of Ag NPs were characterized by using FlFFF, zeta potentiometer, and TEM technique. The mean particle sizes of Ag NPs as characterized by FlFFF were 9 nm, 19 nm, and 45 nm for citrate stabilized-, pectin stabilized-, and alginate stabilized-Ag NPs, respectively, in deionized water. Further, FlFFF with ETAAS detection was employed to observe the aggregation of Ag NPs of various types in environmental water in the absence and presence of humic acid. Citrate stabilized-Ag NPs underwent aggregation more rapid than the pectin stabilized- and alginate stabilized-Ag NPs as the latter two types were sterically stabilized. Further, humic acid could prolong the stability of Ag NPs in the environment.     

A comparison of 2-D chromatography separations using UV and (18)O quantification of proteins in similar proteomes

Proteins present in two mitochondria preparations were separated by 2-D chromatography using the ProteomeLab PF-2D Protein Fractional System, protein fractionation in two dimensions (PF-2D). The proteins in each first-dimension fraction were determined by trypsinization and LC-MS/MS. Chromatography peaks were quantified by UV detection using the "Mapping Tools" software (Beckman). The proteins present in UV detected peaks were trypsinized and identified by automated MS/MS sequencing. Relative amounts of the proteins present in the equivalent peak for each sample were assessed by comparison of the intensities of the constituent peptides and a predicted PF-2D value was calculated from the total ion count (TIC) for each peptide. Relative quantification for (18)O labeled peptides was performed using the ZoomQuant (v1.43b) software [1, 2]. We found that the chromatography peaks detected by UV generally contained several proteins. Using (18)O labeling we determined that in each peak the ratios of the constituent proteins were different. When these ratios were normalized using the TIC to account for abundance, the resulting ratio corresponded to that determined by UV. The predicted value for the PF-2D score corresponded to the observed value for each peak irrespective of the number or proteins detected.     

Proteomic Analysis of Differentially Expressed Proteins of Arabidopsis thaliana Response to Specialist Herbivore Plutella xylostella

The changes of the proteome in Arabidopsis thaliana leaves were examined by specialist Plutella xylostella.Analysis of about 1100 protein spots on each 2DE gel revealed 38 differentially expressed protein spots in abun-dance of which 34 proteins were identified by MALDI-TOF/TOF MS.Among the insect feeding responsive proteins,a few proteins involved in carbon metabolism were identified including proteins associated with the Calvin cycle in the chloroplast and TCA cycle in the mitochondria,indicating carbon metabolism related proteins may play crucial roles in induced defense response in plants under insect infestation.The analysis elucidates the subcellular location of proteins demonstrates that about 50% of proteins are in the chloroplast,which shows the chloroplast has a key role in the insect feeding response for plant.Gene expression analysis of 10 different proteins by quantitative real-time PCR shows that four proteins of the mRNA level were correlated well with the protein level.This study further dissected the nature of insect infestation as a stress signal and some novel insect feeding responsive proteins identified may play an important role in induced defence machanism for plant.     

A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome

For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The “GELFrEE” (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5–25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.