Plasma-derived clotting factor VIII: heterogeneity evaluation in the quest for potential inhibitory-antibody stimulating factors

In this study, we investigated the heterogeneity and the purity grade of three commercially available plasma‐derived clotting factor VIII (FVIII) concentrates, which highly differ with regard to purification strategies, relative concentrations of stabilizers (von Willebrand factor, with or without albumin) and virus inactivation strategies (solvent/detergent and/or heat/pasteurization treatments). Western blot analyses were used to evaluate product‐specific variations from Emoclot^®, Alphanate^® and Haemate^® both in the presence and absence of reducing agents (dithiotreithol). All the plasma‐derived concentrates showed a strong heterogeneity, as they all included a significant amount of truncated forms of the full‐length (FL) clotting FVIII protein. The intact protein accounted for the 38% of the total FVIII proteins in Haemate^® and 29 and 23% in Alphanate^® and Emoclot^®, respectively. Lower intact FVIII amounts in Emoclot might be mainly due to the low von Willebrand factor dosage and the absence of albumin. Upon addition of thrombin, both the FL and truncated forms of the FVIII protein were almost completely digested. Indeed, after thrombin activation, we could still observe a mixture of B‐domain truncated forms of the FL protein along with biologically active digested‐A1 forms. Batch‐to‐batch variation was tested with no evident changes appearing among different batches. Despite the variables in manufacturing processes, inter‐product comparisons yielded similar results for all the plasma‐derived FVIII considered in this study. However, we could individuate in Emoclot a band that was not digested by thrombin, which we could characterize as the 200 kDa FVIII heavy chain. This investigation prompts new concerns about the strong heterogeneity observed upon thrombin digestion of plasma‐derived FVIII, which might contribute to the development of inhibitory antibodies at an early stage of therapy, and to which extent these untoward phenomena could be avoided through direct intervention on routine manufacturing processes.     

Native analysis of plasma-derived clotting factor VIII concentrates: "sponge effect" and contaminants

In the present study, we analysed two commercially available plasma-derived FVIII preparations, Beriate and Emoclot, through native gel-based approaches (CN-PAGE). The rationale behind this study was to assess whether protein complexes from plasma resisted the aggressive manufacturing processes. A preliminary analysis of plasma complexes was performed focusing on the molecular weight range between 45 kDa and 1 MDa. As a result, we could evidence in both preparations the presence of complexes, which resisted the cryoprecipitation, chromatographic and heat treatment processing steps. These complexes behaved in a "sponge-effect"-like fashion through the enrichment of otherwise less abundant contaminants. The use of an alternative electrophoretic approach such as CN-PAGE allowed us to confirm 2DE-based observations and expand the list of non-FVIII proteins which contaminate the preparations.     

Proteomic characterization of plasma‐derived clotting factor VIII–von Willebrand factor concentrates

Proteomic methods were used to identify the levels of impurities in three commercial plasma‐derived clotting factor VIII‐von Willebrand factor (FVIII/VWF) concentrates. In all three concentrates, significant amounts of other plasma proteins were found. In Octanate and Haemoctin, two concentrates developed in the 1990s, the major impurities identified were inter‐α inhibitor proteins, fibrinogen and fibronectin. These two concentrates were also found to contain additional components such as clotting factor II (prothrombin) that are known activators of FVIII. In Wilate, a recently developed FVIII/VWF concentrate, the amount of these impurities was significantly reduced. Batch‐to‐batch variations and differences between three investigated products were detected using iTRAQ, an isotope labeling technique for comparative MS, demonstrating the potential value of this technique for quality control analysis. The importance of thorough proteomic investigations of therapeutic FVIII/VWF preparations from human plasma is also discussed.     

Evaluation of extraction procedures for 2‐DE analysis of aphid proteins

Protein sample preparation is a crucial step in a 2‐DE proteomics approach. In order to establish a routine protocol for the application of proteomics analysis to aphids, this study focuses on the specific protein extraction problems in insect tissues and evaluates four methods to bypass them. The approaches of phenol extraction methanol/ammonium acetate precipitation (PA), TCA/acetone precipitation, PEG precipitation, and no precipitation were evaluated for proteins isolation and purification from apterous adult aphids, Sitobion avenae. For 2‐DE, the PA protocol was optimal, resulting in good IEF and clear spots. PA method yielded the greatest amount of protein and displayed most protein spots in 2‐DE gels, as compared with the TCA/acetone precipitation, PEG precipitation and no precipitation protocols. Analysis of protein yield, image quality and spot numbers demonstrate that the TCA/acetone precipitation protocol is a reproducible and reliable method for extracting proteins from aphids. The PEG precipitation approach is a newly developed protein extraction protocol for aphids, from which more unique protein spots can be detected, especially for detection of acid proteins. These protocols are expected to be applicable to other insects or could be of interest to laboratories involved in insect proteomics, despite the amounts and types of interfering compounds vary considerably in different insects.     

In‐gel equilibration for improved protein retention in 2DE‐based proteomic workflows

The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second‐dimension SDS‐PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in‐solution‐based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second‐dimension SDS‐PAGE gel, referred to as in‐gel equilibration. We show that in‐gel equilibration is effective at reduction and alkylation in SDS‐PAGE gels. Quantification of whole‐cell extracts separated on 2DE gels shows that in‐gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.     

Sugarcane proteomics: Establishment of a protein extraction method for 2‐DE in stalk tissues and initiation of sugarcane proteome reference map

Sugarcane is an important commercial crop cultivated for its stalks and sugar is a prized commodity essential in human nutrition. Proteomics of sugarcane is in its infancy, especially when dealing with the stalk tissues, where there is no study to date. A systematic proteome analysis of stalk tissue yet remains to be investigated in sugarcane, wherein the stalk tissue is well known for its rigidity, fibrous nature, and the presence of oxidative enzymes, phenolic compounds and extreme levels of carbohydrates, thus making the protein extraction complicated. Here, we evaluated five different protein extraction methods in sugarcane stalk tissues. These methods are as follows: direct extraction using lysis buffer (LB), TCA/acetone precipitation followed by solubilization in LB, LB containing thiourea (LBT), and LBT containing tris, and phenol extraction. Both quantitative and qualitative protein analyses were performed for each method. 2‐DE analysis of extracted total proteins revealed distinct differences in protein patterns among the methods, which might be due to their physicochemical limitations. Based on the 2‐D gel protein profiles, TCA/acetone precipitation‐LBT and phenol extraction methods showed good results. The phenol method showed a shift in pI values of proteins on 2‐D gel, which was mostly overcome by the use of 2‐D cleanup kit after protein extraction. Among all the methods tested, 2‐D cleanup‐phenol method was found to be the most suitable for producing high number of good‐quality spots and reproducibility. In total, 30 and 12 protein spots commonly present in LB, LBT and phenol methods, and LBT method were selected and subjected to eLD‐IT‐TOF‐MS/MS and nESI‐LC‐MS/MS analyses, respectively, and a reference map has been established for sugarcane stalk tissue proteome. A total of 36 nonredundant proteins were identified. This is a very first basic study on sugarcane stalk proteome analysis and will promote the unexplored areas of sugarcane proteome research.     

Efficient separation of high‐purity compounds from Oxytropis falcata using two‐dimensional preparative chromatography

The separation of high‐purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two‐dimensional reversed‐phase preparative method was successfully developed for the separation of high‐purity compounds from Oxytropis falcata . Based on the analysis results, an ODS C18 prep column was used for first‐dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high‐purity compounds in the second‐dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans‐cinnamic acid ( 1 ) and trifolirhizin ( 2 ) from Oxytropis falcata . This method has the potential to be an efficient separation method of high‐purity compounds from Oxytropis falcata and it shows great promise for the separation of high‐purity compounds from complex samples.     

Delta2D and Proteomweaver: Performance evaluation of two different approaches for 2‐DE analysis

2‐DE is a fundamental technology used in proteomics research. However, despite its high capacity to simultaneously separate several proteins for subsequent identification and quantitative comparison studies, a drawback for this technique is its limited reproducibility, especially when comparing data from different laboratories. 2‐DE‐related variability can be broadly divided into two categories: experimental and post‐experimental. Experimental variability depends on physical and chemical parameters, whereas post‐experimental variability arises when gels are analyzed by different software packages, particularly when different workflows are followed. In this paper, we compared the analysis performance of two software packages, Delta2D and Proteomweaver, using both standard and experimental gel images. Using standard gel images, the false negative spot count was 50% lower, the false positive count was 77% lower, the true positive count was 19% higher and spot matching was 4% higher in Delta2D when compared to Proteomeweaver. Using experimental gel images, we found that the total amount of time taken to complete the analysis with Delta2D was 30% that of the time needed with Proteomweaver and required fewer user interventions. The differences between ease of use and workflow strategy of these programs is discussed.     

Large‐scale isolation of high‐purity anthocyanin monomers from mulberry fruits by combined chromatographic techniques

Anthocyanins have attracted attention over the past several decades because of their beneficial health effects. In this research, a strategy combining column chromatography and high‐speed countercurrent chromatography was developed for the separation of high‐purity anthocyanin monomers from mulberry fruits. After purification using Amberlite XAD‐7HP column with 80% ethanol (0.1% HCl), a fraction of anthocyanins mixtures with a purity of 68.6% was obtained. High‐speed countercurrent chromatography with a biphasic solvent system of n‐butanol/methyl tert‐butyl ether/acetonitrile/water/trifluoroacetic acid (30:10:10:50:0.05, v/v) was used to separate the anthocyanin monomers. Three monomers of delphinidin‐3‐O‐ rutinoside, cyanidin‐3‐O‐ rutinoside, and cyanidin‐3‐O‐ glucoside were obtained, and identified by ¹H and ¹³C NMR spectroscopy and high‐performance liquid chromatography with electrospray ionization‐mass spectrometry. The method developed in this work can be used to conduct large‐scale separations of anthocyanin monomers from mulberry fruits and other plants.     

Separation of high‐purity eicosapentaenoic acid and docosahexaenoic acid from fish oil by pH‐zone‐refining countercurrent chromatography

The evidence for unique effects of eicosapentaenoic acid and docosahexaenoic acid is growing. Further understanding and exploration of their independent effects in the nutraceutical and pharmaceutical industry is calling for the more efficient separation techniques to overcome the equivalent chain length rule of fatty acids. In this study, free eicosapentaenoic and docosahexaenoic acid were successfully separated by pH‐zone‐refining countercurrent chromatography for the first time. The different solvent systems and the influence of retainer and eluter concentration on the separation efficiency were investigated. A two‐phase solvent system composed of n‐heptane/methanol/water (100:55:45, v/v) was selected with 50 mM of trifluoroacetic acid as retainer in the organic phase and 40 mM of ammonium hydroxide as an eluter in the aqueous phase for the separation of 500 mg of free fatty acids from a refined fish oil sample. 79.6 mg of eicosapentaenoic acid and 328.3 mg docosahexaenoic acid were obtained with the purities of 95.5 and 96.9% respectively determined by gas chromatography with mass spectrometry after methyl esterification. The scale‐up separation of 1 g of samples from both refined and crude fish oil after urea complexation were also achieved successfully with a markedly increased concentration 150 mM of retainer, producing satisfactory yields and purities of targets.     

Proteomics of cypress pollen allergens using double and triple one-dimensional electrophoresis

Italian cypress (Cupressus sempervirens, Cups) pollen causes allergic diseases in inhabitants of many of the cities surrounding the Mediterranean basin. However, allergens of Cups pollen are still poorly known. We introduce here a novel proteomic approach based on double one‐dimensional gel electrophoresis (D1‐DE) as an alternative to the 2‐DE immunoblot, for the specific IgE screening of allergenic proteins from pollen extracts. The sequential one‐dimensional combination of IEF and SDS‐PAGE associated with IgE immunoblotting allows a versatile multiplexed immunochemical analysis of selected groups of allergens by converting a single protein spot into an extended protein band. Moreover, the method appears to be valuable for MS/MS identification, without protein purification, of a new Cups pollen allergen at 43 kDa. D1‐DE immunoblotting revealed that the prevalence of IgE sensitization to this allergen belonging to the polygalacturonase (PG) family was 70% in tested French allergic patients. In subsequent triple one‐dimensional gel electrophoresis, the Cups pollen PG was shown to promote lectin‐based protein‐protein interactions. Therefore, D1‐DE could be used in routine work as a convenient alternative to 2‐DE immunoblotting for the simultaneous screening of allergenic components under identical experimental conditions, thereby saving considerable amounts of sera and allergen extracts.     

Combinatorial peptide ligand library plasma treatment: Advantages for accessing low‐abundance proteins

Depletion of major blood proteins is one of the most promising approaches to accessing low‐abundance biomarkers. This study compared the use of combinatorial peptide ligand library (CPLL) and albumin and immunoglobulins (IgGs) depletion technology for accessing these low‐abundance proteins in plasma using 2‐DE in an acidic restricted pH range (4–7). Compared with native plasma, both techniques enlarge the visibility of other proteins than albumin and IgG, but there were marked differences in their composition. An increase of the number of spots was detected compared with native plasma (157 spots) with 427 and 557 spots, respectively, detected with albumin and IgG depletion, and CPLL treatment. We selected 70 spots to be identified by MALDI‐TOF related to their absence in the 2‐D gels from native or albumin and IgG‐depleted plasma. The 42 spots identified corresponded to 24 different proteins, with more than half of the proteins which did not belong to the major plasma proteins. CPLL treatment allowed the accessibility to proteolytic fragments obtained from major plasma proteins. We found a large superiority of the CPLL approach over the albumin and IgG depletion process. These findings show the utility of depleting major blood proteins to be able to access low‐abundance proteins and the potential of CPLL to select and identify candidate biomarkers in clinical studies.     

Theoretical analysis of vibrational spectra and scaling‐factor of 2‐aryl‐1,3,4‐oxadiazole derivatives

In this study, the direct molecular structure implementations for calculating vibrational spectra and scaling factors, and infrared intensities at both the Hartree–Fock (HF) and density functional (B3LYP) levels of theory with 6‐31G(d), 6‐311G(d), 6‐31++G(d,p), and 6‐311++G(d,p) basis sets are presented. Also, vibrational frequencies have been investigated as dependence on the choice of method and basis set. The parameters of molecular geometry and vibrational frequencies values of 2‐aryl‐1,3,4‐oxadiazoles 5a–g in the ground state have been calculated. Theoretical determination of vibrational frequencies is quite useful both in understanding the relationship between the molecular structures and scaling factor. The data of 2‐aryl‐1,3,4‐oxadiazoles 5a–g display significant electronic properties provide the basis for future design of efficient materials having the oxadiazole core and theoretical IR studies. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Efficient purification of high‐purity compounds from the stem of Lonicera japonica Thunb using two‐dimensional preparative chromatography

Purification of high‐purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two‐dimensional preparation method was successfully developed for the preparation of high‐purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first‐dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high‐purity compounds in the second‐dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high‐purity compounds from complex samples.     

An expedient reverse‐phase high‐performance chromatography (RP‐HPLC) based method for high‐throughput analysis of deferoxamine and ferrioxamine in urine

The present study was planned to optimize and validate an expedient reverse‐phase high chromatography (RP‐HPLC) based protocol for the analysis of deferoxamine (DFO) and ferrioxamine (FO) in urinary execration of patients suffering β ‐thalassemia major. The optimized RP‐HPLC method was found to be linear over the wide range of DFO and FO concentration (1–90 μg/mL) with appreciable recovery rates (79.64–97.30%) of quality controls at improved detection and quantitation limits and acceptable inter and intraday variability. Real‐time analysis of DFO and FO in the urine of thalassemic patients (male and female) at different intervals of Desferal®(Novartis Pharmaceuticals Corporation) injection revealed DFO and FO excretion at significantly (p < 0) different rates. The maximum concentrations of DFO (76.7 ± 3.06 μg/mL) and FO (74.2 ± 3.25 μg/mL) were found in urine samples, collected after 6 h of drug infusion while the minimum levels of DFO (1.10 ± 0.12 μg/mL) and FO (2.97 ± 0.13 μg/mL) were excreted by patients after 24 h. The present paper offers balanced conditions for an expedient, reliable and quick determination of DFO and FO in urine samples.     

VinaMPI: Facilitating multiple receptor high‐throughput virtual docking on high‐performance computers

The program VinaMPI has been developed to enable massively large virtual drug screens on leadership‐class computing resources, using a large number of cores to decrease the time‐to‐completion of the screen. VinaMPI is a massively parallel Message Passing Interface (MPI) program based on the multithreaded virtual docking program AutodockVina, and is used to distribute tasks while multithreading is used to speed‐up individual docking tasks. VinaMPI uses a distribution scheme in which tasks are evenly distributed to the workers based on the complexity of each task, as defined by the number of rotatable bonds in each chemical compound investigated. VinaMPI efficiently handles multiple proteins in a ligand screen, allowing for high‐throughput inverse docking that presents new opportunities for improving the efficiency of the drug discovery pipeline. VinaMPI successfully ran on 84,672 cores with a continual decrease in job completion time with increasing core count. The ratio of the number of tasks in a screening to the number of workers should be at least around 100 in order to have a good load balance and an optimal job completion time. The code is freely available and downloadable. Instructions for downloading and using the code are provided in the Supporting Information. © 2013 Wiley Periodicals, Inc.     

Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.     

Shapeable matrix‐free Spectra® fiber‐reinforced polymeric composites via high‐temperature high‐pressure sintering: Process‐structure‐property relationship

In an exploratory effort to find a new way to make high‐performance composites used in ballistic protective applications, matrix‐free Spectra® fiber‐reinforced polymeric composites are produced via a novel processing method called high‐temperature high‐pressure sintering. Mechanical testing at ambient and elevated temperatures proves that the fibers can maintain their properties after processing. The characteristics and properties of the final products vary with different processing conditions. Their microstructure and morphology were investigated using SEM and WAXD. Their mechanical properties, including interlayer adhesion, rigidity, and ballistic performance, were measured and compared with those of the conventional composites. The sintering mechanism is proposed and verified. Spectra cloth is capable of being shaped to produce complex double curvatures by a thermoforming process, using a simple hemispherical mold. Success in different molding sequences and procedures shows the versatility in manufacturing. The theoretical background for the thermoformability is explained in terms of molecular interaction, microstructure, and morphology. Selective thermomechanical properties of the molded structures were measured. By combining the knowledge and information from the aforementioned studies, the process‐structure‐property relationship is established, which gives in‐depth and better understanding of this unique high‐temperature high‐pressure sintering process for consolidating Spectra cloth. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 2767–2789, 2005