Combination of affinity depletion of abundant proteins and reversed-phase fractionation in proteomic analysis of human plasma/serum

The tremendous complexity of the serum and plasma proteome presents extreme analytical challenges in comprehensive analysis due to the wide dynamic range of protein concentrations. Therefore, robust sample preparation methods remain one of the important steps in the proteome characterization workflow. We present the results on a new column for the specific depletion of 14 high-abundant proteins from human serum and plasma and the subsequent reversed-phase fractionation of the flow-through proteins. Analysis of tryptic peptides was accomplished with microfluidic HPLC-Chip/MS system. Results indicate that high-abundant protein depletion combined with RP fractionation of plasma showed an improved dynamic range for proteomic analysis and enabled the identification of low-abundant plasma proteins.     

Structural,functional, and evolutionary analysis of late embryogenesis abundant proteins (LEA) in Triticum aestivum: A detailed molecular level biochemistry using in silico approach

LEA (Late Embryogenesis Abundant) proteins are abundant in plants and play a crucial role in abiotic stress tolerance. In our work, we primarily focused on the variations in physiochemical properties, conserved domains, secondary structure, gene ontology and evolutionary relationships among 40 LEA proteins of Triticum aestivum (common wheat). Wheat LEA protein belongs to first 6 classes out of the 13 classes present in LEApdB, the comprehensive database for LEA proteins. Proteins belonging to each LEApdB class have structures and functions distinguished from other classes. The study found three different conserved LEA domains in Triticum aestivum. One important domain was dehydrin, present in wheat proteins of classes 1, 2 and 4, though varied in sequence level, have similar biological processes. The study also found sequence level and phylogenetic similarity between dehydrin domains of class 1 and 4, but distinct from that of LEApdB class 2. This study also demonstrated functional diversity in two class 6 proteins occurred due to many destabilizing mutations in the LEA4 domain that caused alteration of ligand binding and conformational shift from 3₁₀-helix → turn within the domain. The LEA4 domains of these proteins also showed functional similarity and evolutionary relatedness with three other proteins of genus Aegilops, denoting that these proteins in Triticum aestivum were derived from its ancestor Aegilops. The study also assigned LEApdB class 4 to an unclassified LEA protein ‘WZY2-1’ based on amino acid composition, conserved domain, motif architecture and phylogenetic relatedness with class 4 proteins. Our study has revealed a detailed analysis of LEA proteins in Triticum aestivum and can serve as a pillar for further investigations and comparative analysis of wheat LEA proteins with other cereal or plant types.     

A new procedure using membrane chromatography for the valorization of fraction IV from Kistler and Nitschmann's fractionation of blood plasma

Summary The aim of this work was to recover albumin from Kistler and Nitschmann's fraction IV using membrane chromatography. The best solubilization results were obtained at pH 6.5 using a phosphate buffer containing 150 mM NaCl. More than 90% of the initial albumin content was recovered.The purification procedure included 2 main steps: the first one was a depth filtration in order to remove fine particles and a selective depth filter treatment for lipid removal. The second step was ion exchange chromatography. We used membrane chromatography systems where the fluid flows radially allowing fast flow purification under low operating pressures. The eluted albumin was free from IgG. Because of the absence of contaminating IgG and its high microbiological quality compared to standard animal sera, this albumin preparation can be used as a culture medium additive. It can also be further purified by ultrafiltration. The equipment used here is easy to handle and to sterilize, and meets the FDA Code of Federal Regulations. Additionally, this procedure is flexible enough to allow the co-purification of other fraction IV proteins such as transferrin or alpha-1-anti-trypsin.     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

A study of the interactions between carboplatin and blood plasma proteins using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry

To study the carboplatin–protein interaction, a sensitive method using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC–ICP–MS) was developed. The complexes formed between plasma proteins and carboplatin were monitored and identified with this method. Composite blood plasma samples from patients who were undergoing chemotherapy were analyzed, and carboplatin was found to bind plasma proteins. In addition, blank plasma samples were spiked with carboplatin and were analyzed as a time course study, and the results confirmed that carboplatin formed complexes with plasma proteins, primarily albumin and γ-globulin. To further substantiate the study, these two proteins were incubated with carboplatin. The binding between carboplatin and these proteins was then characterized qualitatively and quantitatively. In addition to a one-to-one binding of Pt to protein, protein aggregation was observed. The kinetics of the binding process of carboplatin to albumin and γ-globulin was also studied. The initial reaction rate constant of carboplatin binding to albumin was determined to be 0.74 M⁻¹ min⁻¹, while that for γ-globulin was 1.01 M⁻¹ min⁻¹, which are both lower than the rate constant of the cisplatin–albumin reaction previously reported.     

Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries

The affinity inhibitor of fusion peptide of influenza A virus has been studied using a combination of high-performance affinity chromatography (HPAC) and combinatorial peptide libraries. Fusion peptide (FP) (1-11) of influenza A virus was used as the affinity ligand and immobilized onto the poly(glycidyl methacrylate) (PGMA) beads. Positional scanning peptide libraries based on antisense peptide strategy and extended peptide libraries were designed and synthesized. The screening was carried out at acidic pH (5.5) in order to imitate the environment of virus fusion. A hendecapeptide FHRKKGRGKHK was identified to have a strong affinity to the FP (1-11). The dissociation constant of the complex of the hendecapeptide and the FP (1-11) is 3.10 x 10(-6) mol l(-1) in a physiological buffer condition. The polypeptide has a fairly inhibitory effect on three different strains of influenza A virus H1N1 subtype.     

Determination of saterinone enantiomers in plasma samples with an internal standard using a Chiralcel OD column, fractionation and reversed-phase chromatography

A specific and validated high-performance liquid chromatographic method was developed for the determination of the S-(-) and R-(+) enantiomers of saterinone. 1-[(4-cyano-1,2-dihydro-6-methyl-2-oxopyridin-5-yl)phenoxyl] -3-[4-(2- methoxyphenyl)piperazin-1-yl]propan-2-ol, in plasma at the low ng/ml level. The enantiomers of saterinone and an internal standard, 1-[(4-cyano-1,2-dihydro-6-methyl-2-oxo-pyridin-5-yl)phenoxy]-3-[4-(2- ethoxyphenyl)piperazin-1-yl]propan-2-ol, were chromatographed on a chiral Chiralcel OD stationary phase. However, the S-(-) enantiomers of saterinone and the internal standard were unresolved, as were the R-(+) enantiomers of both substances. Therefore, the two fractions were collected and each was separately resolved on an achiral Polyencap A reversed-phase column and quantified. The detection limit was 0.5 ng/ml of enantiomer, allowing the determination of plasma levels up to 36 h after oral administration of 90, 150 and 180 mg of saterinone to twelve subjects.     

Application of an improved proteomics method, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry, to differential analysis of proteins in small regions of mouse brain

To identify age-related proteins in small regions of mouse brain, we improved a proteomics approach, fluorogenic derivatization-liquid chromatography-tandem mass spectrometry (FD-LC-MS/MS), and applied the method to the differential proteome analysis of aging in cerebral cortex, hippocampus and brainstem. The method showed good accuracy with RSDs <10% for between-day protein peak heights, and much better sensitivity for the detection of proteins compared to other proteomics approaches. The existence of 28 regionally specific age-related proteins in mouse brain was demonstrated. These results verified that the small brain regions could be the targets for proteome analysis by the FD-LC-MS/MS method.     

Analysis of human blood plasma triacylglycerols using capillary gas chromatography, silver ion thin-layer chromatographic fractionation and desorption chemical ionization mass spectrometry.

The identification and quantitation from pooled human plasma of over 50 molecular species of triacylglycerols, including 22 not previously reported, are reported. The triacylglycerols were first resolved by silver ion thin-layer chromatography into seven fractions, which were independently analysed by polarizable capillary gas chromatography and desorption chemical ionization mass spectrometry in the presence of an internal standard. The two methods gave similar values for the estimates of oligoenoic species, but the former method underestimated the polyenes (due mainly to significant losses caused by thermal degradation), and the latter method overestimated the saturates. The results show that an effective analysis of molecular species of plasma triacylglycerols cannot be obtained by either technique alone.     

Application of ion-exchange and lipophilic-gel chromatography to the purification and group fractionation of steroidal spirolactones, isolated from biological fluids

Chromatography of steroidal spirolactones on DEAE-Sephadex A-25 under selected pH conditions allowed efficient separation of these compounds from other steroids and many of the endogenous components of human urine. The spirolactones were recovered in high yield, mostly over 90%. Lipophilic-gel chromatography provided a useful method for group fractionation of mixtures of these spirolactones with high recoveries (generally over 90%), unaffected by the presence of endogenous material from normal human urine.     

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.     

A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification,enrichment via solid-phase reversible binding,and analysis by mass spectrometry

beta-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO(4)(3-) elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for beta-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available alpha-casein. Enriched peptide fragments were characterized by MALDI-TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.     

A novel two-zone protein uptake model for affinity chromatography and its application to the description of elution band profiles of proteins fused to a family 9 cellulose binding module affinity tag

A novel two-zone model (TZM) is presented to describe the rate of solute uptake by the stationary phase of a sorption-type chromatography column. The TZM divides the porous stationary-phase particle into an inner protein-free core and an outer protein-containing zone where intraparticle transport is limited by pore diffusion and binding follows Langmuir theory. The TZM and the classic pore-diffusion model (PDM) of chromatography are applied to the prediction of stationary-phase uptake and elution bands within a cellulose-based affinity chromatography column designed to selectively purify proteins genetically labelled with a CBM9 (family 9 cellulose binding module) affinity tag. Under both linear and nonlinear loading conditions, the TZM closely matches rates of protein uptake within the stationary phase particles as measured by confocal laser scanning microscopy, while the PDM deviates from experiment in the linear-binding region. As a result, the TZM is shown to provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed.     

A metabonomic analysis of plasma from Zucker rat strains using gas chromatography/mass spectrometry and pattern recognition

Plasma obtained from three strains of Zucker rats was analysed using capillary gas chromatography/mass spectrometry (GC/MS) to obtain global metabolite profiles as part of a series of metabonomic investigations of animal models of diabetes. Samples were obtained from 20-week-old male wild-type Zucker lean, (fa/fa) obese and lean/(fa) animals and were analysed following protein precipitation, using acetonitrile, and derivatisation. Subsequent data analysis using principal components analysis (PCA) and orthogonal projection to latent structures (OPLS) revealed differences between the plasma metabolite profiles of the three strains, with those of the Zucker lean and the lean/(fa) crosses being similar to each other whilst differing from the (fa/fa) obese strain.     

Comprehensive analysis of low-abundance proteins in human urinary exosomes using peptide ligand library technology, peptide OFFGEL fractionation and nanoHPLC-chip-MS/MS

Human urinary exosomes are 30-100 nm vesicles that originate as the internal vesicles in multivesicular bodies from every renal epithelial cell type facing the urinary track and may serve as a suitable noninvasive starting material for biomarker discovery relevant to a variety of renal disease. To comprehensively explore the low-abundance proteome, combinatorial peptide ligand libraries, combined with peptide OFFGEL electrophoresis were employed for the enrichment and separation of relatively low-abundant proteins in urinary exosomes. After analysis by nanoHPLC-chip-MS/MS, 512 proteins were identified, including a large number of proteins with extreme molecular weight or extreme pI value, which could not be well mapped by using traditional 2-D-gel-based separation methods. This in-depth analysis of low-abundant proteins in urinary exosomes led to an increased understanding of molecular composition of these little vesicles and may be helpful for the discovery of novel biomarker. Our work also provides an effective strategy of concentration and identification of low-abundance proteome from complex bio-samples.     

One-step purification of rat plasma alpha 1-acid glycoprotein by antibody affinity chromatography: application to normal and inflamed rat sera

Most purification procedures used previously to isolate alpha 1-acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.     

Determination of afloqualone in human plasma using liquid chromatography/tandem mass spectrometry: Application to pharmacokinetic studies in humans

Two methods for determining the central-acting muscle relaxant afloqualone in human plasma were developed and compared using API2000 and API4000 liquid chromatography tandem mass spectrometry (LC/MS/MS) systems. In the API2000 LC/MS/MS system, afloqualone and the internal standard methaqualone were extracted from plasma using a methyl-tertiary ether. After drying the organic layer, the residue was reconstituted in a mobile phase (0.1% formic acid-acetonitrile:0.1% formic acid buffer, 80:20 v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 117 for afloqualone and methaqualone, respectively.Sample preparation for the API4000 LC/MS/MS system involved simple protein precipitation with an organic mixture (methanol:10% ZnSO₄ = 8:2). The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 131 for afloqualone and methaqualone, respectively.In both assays, the coefficient of variation of the precision was less than 11.8%, the accuracy exceeded 91.5%, the limit of quantification was 0.5 ng/ml, and the limit of detection was 0.1 ng/ml for afloqualone. Two methods were used to measure the plasma afloqualone concentration in healthy subjects after a single oral 20-mg dose of afloqualone. During subsequent application of the methods, we observed that high-concentration plasma samples (>7 ng/ml) prepared using the protein precipitation method resulted in about 20% higher afloqualone concentrations than with plasma samples prepared using the liquid-liquid extraction method. We believe that this phenomenon was related to the cleanness of the sample and its chemical nature.     

Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties

Recent efforts at the proteomic level were employed to describe the protein equipment of the plasma membrane of the model plant Arabidopsis thaliana. These studies had revealed that the plasma membrane is rich in extrinsic proteins but came up against two major problems: (i) few hydrophobic proteins were recovered in two-dimensional electrophoresis gels, and (ii) many plasma membrane proteins had no known function or were unknown in the database despite extensive sequencing of the Arabidopsis genome. In this paper, several methods expected to enrich a membrane sample in hydrophobic proteins were compared. The optimization of solubilization procedures revealed that the detergent to be used depends on the lipid content of the sample. The corresponding proteomes were compared with the statistical model AMMI (additive main effects with multiplicative interaction) that aimed at regrouping proteins according to their solubility and electrophoretic properties. Distinct groups emerged from this analysis and the identification of proteins in each group allowed us to assign specific features to several of them. For instance, two of these groups regrouped very hydrophobic proteins, one group contained V-ATPase subunits, another group contained proteins with one transmembrane domain as well as proteins known to interact with membrane proteins. This study provides methodological tools to study particular classes of plasma membrane proteins and should be applicable to other cellular membranes.     

Quantitative analysis of betulinic acid in mouse,rat and dog plasma using electrospray liquid chromatography/mass spectrometry

Betulinic acid is under development as a therapeutic agent for the treatment of metastatic malignant melanoma. In support of pharmacokinetic and toxicological evaluations, a robust assay based on liquid chromatography/mass spectrometry (LC/MS) was developed for the quantitative analysis of betulinic acid. Sample preparation consisted of deproteinization of the plasma by the addition of three volumes of acetonitrile and one volume of methanol followed by centrifugation. Aliquots of the supernatant were analyzed using an isocratic reversed-phase high-performance liquid chromatography (HPLC) system coupled to a negative ion electrospray mass spectrometer. Deprotonated molecules of betulinic acid and the isomeric internal standard oleanolic acid were detected using selected ion monitoring at m/z 455. The limit of detection of betulinic acid was 0.5 pg (1.1 fM) injected on-column (50 pg/mL, 10 microL injection volume), and the limit of quantitation was 2 pg (4.4 fM, 200 pg/mL, 10 microL injection volume). Betulinic acid was stable in plasma samples at -20 degrees C for at least 3 weeks. The intra-day and inter-day coefficients of variation of the assay were < or =6.4 and < or =9.0%, respectively. The utility of the assay was demonstrated by analyzing betulinic acid spiked into mouse, rat and dog plasma, by determining the extent of binding of betulinic acid to plasma proteins, and by measuring betulinic acid in mouse and rat plasma following intraperitoneal or intravenous administration in vivo. At 15 and 25 microg/mL in mouse, rat or dog plasma, betulinic acid was 99.99% bound to serum proteins, and, at 5 microg/mL, betulinic acid was > or =99.97% bound.     

A novel approach to the rapid determination of amoxicillin in human plasma by solid phase microextraction and liquid chromatography

A new approach to the rapid determination of amoxicillin (AMO) in human plasma followed by solid phase microextraction (SPME) fiber coatings based on conducting polymers (polypyrrole and polythiophene) and high performance liquid chromatography (HPLC) has been described. The porous structures of the electrochemically deposited polymer coatings have been characterized by scanning electron microscopy (SEM). The experimental parameters relating to the extraction efficiency of the SPME fibers such as pH, extraction time and desorption conditions (solvents, time) were studied and selected. The SPME/HPLC-UV method was linear over a working range of 1-50 μg ml(-1). The inter-day accuracy (expressed as coefficients of variations, CVs) was less than 15% and precision (expressed as the relative standard deviations, RSDs) with percentage values was less than 5.9%. Amoxicillin was found to be stable in the human plasma at room temperature (20 °C) within 8 hours. The developed method was successfully applied to the analysis of real human plasma samples. The limit of detection and limit of quantification for amoxicillin in plasma were 1.21 μg ml(-1) and 3.48 μg ml(-1), respectively.