Protein Peak Research Articles

Automated 2D-HPLC method for characterization of protein aggregation with in-line fraction collection device.

Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing. The formation of protein aggregates is one such quality attribute that can lead to safety and efficacy issues in the final drug product. Described in this study is a fully automated two-dimensional high performance liquid chromatography (2D-HPLC) method for characterizing protein aggregation of crude in-process bioreactor samples. It combines protein A purification and separation by size exclusion into a single analytical module that has the potential to be employed at-line within a bioprocessing system. This method utilizes a novel in-line fraction collection device allowing for the collection of up to twelve fractions from a single sample or peak which facilitates the subsequent linked analysis of multiple protein peaks of interest in one chromatography module.

Prognostic prediction by liver tissue proteomic profiling in patients with colorectal liver metastases.

To obtain proteomic profiles in patients with colorectal liver metastases (CRLM) and identify the relationship between profiles and the prognosis of CRLM patients. Prognosis prediction (favorable or unfavorable according to Fong's score) by a classification and regression tree algorithm of surface-enhanced laser desorption/ionization TOF-MS proteomic profiles from cryopreserved CRLM (patients) and normal liver tissue (controls). The protein peak 7371 m/z showed the clearest differences between CRLM and control groups (94.1% sensitivity, 100% specificity, p < 0.001). The algorithm that best differentiated favorable and unfavorable groups combined 2970 and 2871 m/z protein peaks (100% sensitivity, 90% specificity). Proteomic profiling in liver samples using classification and regression tree algorithms is a promising technique to differentiate healthy subjects from CRLM patients and to classify the severity of CRLM patients.

Interactions between metal ions and the biopolymer in activated sludge: quantification and effects of system pH value

The quantification and effects of system pH value on the interactions between Pb(II) and the biopolymer in activated sludge were investigated. The biopolymer had two protein-like fluorescence peaks (Ex/Em = 280 nm/326–338 nm for peak A; Ex/Em = 220–230 nm/324–338 nm for peak B). The fluorescence intensities of peak B were higher than those of peak A. The fluorophores of both peaks could be largely quenched by Pb(II), and the quencher dose for peak B was about half of that for peak A. The modified Stern-Volmer equation well depicted the fluorescence quenching titration. The quenching constant (Ka) values for both peaks decreased with rising system pH value, and then sharply decreased under alkaline conditions. It could be attributed to that the alkaline conditions caused the reduction of available Pb(II) due to the occurrence of Pb(OH)2 sediments. The Ka values of peak B were bigger than those for peak A at the same system pH values. Accordingly, the aromatic proteins (peak B) played a key role in the interactions between metal ions and the biopolymer.

Identification of beta-nerve growth factor in dromedary camel seminal plasma and its role in induction of ovulation in females

The main objective of this study was to demonstrate the effect of seminal plasma ovulation inducing factor (OIF) on ovulation in female camels. Seminal plasma was fractionated using gel filtration chromatography, and two protein peaks were obtained and analysed by western blotting. The effect of the bioactive protein fraction (OIF/β-NGF) was tested by intramuscular injection (1 ml) in synchronized females with the following treatments: PBS (negative control group, n = 3); 20 µg Buserelin (GnRH analogue, positive control group; n = 3); and purified OIF with doses of 1 and 2 mg (n = 5, each treatment). Blood samples were collected every two days from day 0 until 14 days post-treatment, and the progesterone concentration was assessed. The obtained results showed that the OIF is highly present in the seminal plasma of dromedary camels as a protein with a molecular mass of approximately 14 kDa. It was detected as a beta-Nerve Growth Factor, named Cam-β-NGF. The effect of this molecule on ovulation was clearly demonstrated by the significant increase of the plasma progesterone concentration in the treated female groups (1 and 2 mg of Cam-β-NGF) as observed in Buserelin group. In summary, intramuscular injection of β-NGF isolated from dromedary camel seminal plasma induces ovulation in females with similar rate to Buserelin treatment.

Commentary on clinical utility of whole genome sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

3The present study is commentary and aims to evaluate the practical application of Whole Genome Sequencing (WGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in clinical microbiology. The MALDI-TOF MS method has been replaced cultural and biochemical tests for species identification in most laboratories worldwide. Moreover, WGS has elevated the discriminatory power by detection of single nucleotide polymorphisms (SNPs), and helped with the identification of microorganisms that were misidentified by previous culture and biochemical tests. Moreover, WGS can be used in epidemiological studies (e.g. clinical outbreaks) by tracking of phylogenetic maps and analyzing SNP distance; antimicrobial resistance studies such as the study of antimicrobial resistance genes, e.g. CTX-M, NDM, KPC, or OXA; and the study of recombination of plasmid or insertion sequence (IS) elements to elucidate the mechanism of antimicrobial resistance. MALDI-TOF technology is used for bacterial or fungal species identification in most laboratories. VITEK MS (bioMerieux) and Biotyper (Bruker Daltonics) are examples of commercially available MALDI-TOF MS systems. The technique has been used for direct identification of organisms from blood culture bottles, markedly shortening the identification time. Beta-lactamases or carbapenemases have been detected by analyzing specific protein peaks using MALDI-TOF MS. Shiga toxin-producing Escherichia coli, Salmonella serotype, and Vibrio phenotypes can also be detected.

Cranberry Proanthocyanidins - Protein complexes for macrophage activation.

In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas the PAC-HEL complex had already reached the maximum IL-2 expression. Cranberry PAC may increase the rate of endocytosis of HEL and subsequent expression of IL-2 by the T-cell hybridomas. These results suggest that PAC-protein complexes modulate aspects of innate and acquired immune responses in macrophages.

Extraction and purification of protease inhibitor(s) from seeds of Helianthus annuus with effects on Leptinotarsa decemlineata digestive cysteine protease

Abstract The aim of the current investigation was to purify cysteine protease inhibitors from sunflower ( Helianthus annuus L. cv. Record) (Asteraceae) seeds with potential activity on gut protease of Colorado potato beetle (CPB), Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Ammonium sulfate precipitated proteinaceous fractions; 0–30%, 30–50%, 50–70%, and 70–100% showed 41.03%, 48.66%, 41.53%, and 28.83% inhibition on the last instar larval gut general protease activity, respectively. Also, fraction 30–50% showed the highest inhibitory effect on digestive general protease activity of all developmental stages. This fraction was purified using two different chromatography techniques; ion-exchange using DEAE and affinity using SiO 2 -CPB larval gut enzyme mix. Four peaks of protein were eluted from ion-exchange chromatography using NaCl step gradient. When used Z-Ala-Arg-Arg-4msNA as cysteine protease substrates, the purification fold of first fraction was obtained 28.31, also the yield was 66.89%. Fraction of affinity chromatography obtained 26.98 purification fold and yielded 53.96%. Both of the purified protein fractions from two methods showed two similar bands in SDS-PAGE with apparent molecular mass of 14 and 15 kDa. Consequently, the high level of purification fold and yield suggest that both methods were appropriate for purification, but affinity was more specific because CPB gut enzyme was used as ligand.

Identification of Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry as Predictors of Prognosis in Triple Negative Breast Cancer

Triple-negative breast cancer (TNBC) is characterized by lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expressions. Prognosis of TNBC is poor due to recurrence and cancer death even with complete tumor resection, and it remains unclear which biomarkers are clinically useful for prediction of recurrence. The aim of this study was therefore to identify biomarkers for prognosis of TNBC by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). 51 cases of TNBC were collected, including 17 patients with recurrence or death and 34 patients with non-recurrence. Factors that were significantly associated (P < 0.05) with TNBN prognosis after statistical analysis included tumor size, menopausal status and lymph node metastasis. Seven statistical significant peaks were found after protein mass spectrometry, with corrected P < 0.05. The features were selected to predict the patient's outcome using Support Vector Machine (SVM) classifier. When all the peaks were combined to classify the two groups, we obtained high classification accuracy, sensitivity and specificity, which were 90.2%, 82.35% and 94.12%, respectively. The results indicated that the identified protein peaks are potential biomarkers for predicting the prognosis of TNBC patients.

Corrigendum: Detection of Isoforms Differing by a Single Charge Unit in Individual Cells.

During the organization of the final layout, the x-axes in Figure 3 b and 1 e of this Communication were mislabeled. The authors have verified that all of the calculations and findings of the work remain valid. The corrected figure is included below, with the following corrections: scIEF with immunoprobing resolves proteoforms in individual mammalian cells. a) False-color fluorescence micrographs and traces show scIEF detection by immunoprobing of β-TUB and tGFP from individual cells. Microwells are outlined with a black circle, located at 0 mm. Arrows indicate protein peaks; plotted black outlines are the Gaussian fits for identified peaks. b) False-color fluorescence micrographs and traces show detection of denatured β-TUB isoforms in 3 of 8 cells. c) Median total tGFP probing fluorescence (area under the curve, AUC) under native (N) and denaturing (D) conditions (nD=8, nN=9, p<0.01). (d) Relative isoform fractions (nβ-TUB=3, ntGFP=8). e) False-color fluorescence micrographs and traces show detection of lamin A/C from individual cells under denaturing conditions (nD=9). pH range, 4–7; microwell position, 6.75 mm from the catholyte-side edge of the bottom gel in all separations. The authors sincerely apologize for the mislabeled axes.

Prediction and validation of residual feed intake and dry matter intake in Danish lactating dairy cows using mid-infrared spectroscopy of milk

The present study explored the effectiveness of Fourier transform mid-infrared (FT-IR) spectral profiles as a predictor for dry matter intake (DMI) and residual feed intake (RFI). The partial least squares regression method was used to develop the prediction models. The models were validated using different external test sets, one randomly leaving out 20% of the records (validation A), the second randomly leaving out 20% of cows (validation B), and a third (for DMI prediction models) randomly leaving out one cow (validation C). The data included 1,044 records from 140 cows; 97 were Danish Holstein and 43 Danish Jersey. Results showed better accuracies for validation A compared with other validation methods. Milk yield (MY) contributed largely to DMI prediction; MY explained 59% of the variation and the validated model error root mean square error of prediction (RMSEP) was 2.24kg. The model was improved by adding live weight (LW) as an additional predictor trait, where the accuracy R2 increased from 0.59 to 0.72 and error RMSEP decreased from 2.24 to 1.83kg. When only the milk FT-IR spectral profile was used in DMI prediction, a lower prediction ability was obtained, with R2=0.30 and RMSEP=2.91kg. However, once the spectral information was added, along with MY and LW as predictors, model accuracy improved and R2 increased to 0.81 and RMSEP decreased to 1.49kg. Prediction accuracies of RFI changed throughout lactation. The RFI prediction model for the early-lactation stage was better compared with across lactation or mid- and late-lactation stages, with R2=0.46 and RMSEP=1.70. The most important spectral wavenumbers that contributed to DMI and RFI prediction models included fat, protein, and lactose peaks. Comparable prediction results were obtained when using infrared-predicted fat, protein, and lactose instead of full spectra, indicating that FT-IR spectral data do not add significant new information to improve DMI and RFI prediction models. Therefore, in practice, if full FT-IR spectral data are not stored, it is possible to achieve similar DMI or RFI prediction results based on standard milk control data. For DMI, the milk fat region was responsible for the major variation in milk spectra; for RFI, the major variation in milk spectra was within the milk protein region.

Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation.

The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren't detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.

Heavy Sugar and Heavy Water Create Tunable Intact Protein Mass Increases for Quantitative Mass Spectrometry in Any Feed and Organism.

Stable isotope labeling techniques for quantitative top-down proteomics face unique challenges. These include unpredictable mass shifts following isotope labeling, which impedes analysis of unknown proteins and complex mixtures and exponentially greater susceptibility to incomplete isotope incorporation, manifesting as broadening of labeled intact protein peaks. Like popular bottom-up isotope labeling techniques, most top-down labeling methods are restricted to defined media/feed as well as amino acid auxotrophic organisms. We present a labeling method optimized for top-down proteomics that overcomes these challenges. We demonstrated this method through the spiking of 13C-sugar or 2H-water into standard laboratory feedstocks, resulting in tunable intact protein mass increases (TIPMI). After mixing of labeled and unlabeled samples, direct comparison of light and heavy peaks allowed for the relative quantitation of intact proteins in three popular model organisms, including prokaryotic and eukaryotic microorganisms and an animal. This internal standard method proved to be more accurate than label-free quantitation in our hands. Advantages over top-down SILAC include working equally well in nutrient-rich media, conceivably expanding applicability to any organism and all classes of biomolecules, not requiring high-resolving power MS for quantitation and being relatively inexpensive.

Purification and characterization of alginate lyase from locally isolated marinePseudomonas stutzeriMSEA04

An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

Dioxygenases of Chlorobiphenyl-Degrading Species Rhodococcus wratislaviensis G10 and Chlorophenol-Degrading Species Rhodococcus opacus 1CP Induced in Benzoate-Grown Cells and Genes Potentially Involved in These Processes.

Dioxygenases induced during benzoate degradation by the actinobacterium Rhodococcus wratislaviensis G10 strain degrading haloaromatic compounds were studied. Rhodococcus wratislaviensis G10 completely degraded 2g/liter benzoate during 30h and 10g/liter during 200h. Washed cells grown on benzoate retained respiration activity for more than 90 days, and a high activity of benzoate dioxygenase was recorded for 10 days. Compared to the enzyme activities with benzoate, the activity of benzoate dioxygenases was 10-30% with 13 of 35 substituted benzoate analogs. Two dioxygenases capable of cleaving the aromatic ring were isolated and characterized: protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase. Catechol inhibited the activity of protocatechuate 3,4-dioxygenase. Protocatechuate did not affect the activity of catechol 1,2-dioxygenase. A high degree of identity was shown by MALDI-TOF mass spectrometry for protein peaks of the R. wratislaviensis G10 and Rhodococcus opacus 1CP cells grown on benzoate or LB. DNA from the R. wratislaviensis G10 strain was specifically amplified using specific primers to variable regions of genes coding α- and β-subunits of protocatechuate 3,4-dioxygenase and to two genes of the R. opacus 1CP coding catechol 1,2-dioxygenase. The products were 99% identical with the corresponding regions of the R. opacus 1CP genes. This high identity (99%) between the genes coding degradation of aromatic compounds in the R. wratislaviensis G10 and R. opacus 1CP strains isolated from sites of remote location (1400 km) and at different time (20-year difference) indicates a common origin of biodegradation genes of these strains and a wide distribution of these genes among rhodococci.

Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

The effect of supplementing feed with organic selenium on some hematological, biochemical, behavioral markers and egg quality

Non-ischemic cardiomyopathy is a leading cause of congestive heart failure and sudden cardiac death in humans and in some cases the etiology of cardiomyopathy can include the downstream effects of an essential element deficiency. Of all mammal species, pygmy sperm whales (Kogia breviceps) present the greatest known prevalence of cardiomyopathy with more than half of examined individuals indicating the presence of cardiomyopathy from gross and histo-pathology. Several factors such as genetics, infectious agents, contaminants, biotoxins, and inappropriate dietary intake (vitamins, selenium, mercury, and pro-oxidants), may contribute to the development of idiopathic cardiomyopathy in K. breviceps. Due to the important role Se can play in antioxidant biochemistry and protein formation, Se protein presence and relative abundance were explored in cardiomyopathy related cases. Selenium proteins were separated and detected by multi-dimension liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS), Se protein identification was performed by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS), and Se protein profiles were examined in liver (n = 30) and heart tissue (n = 5) by SEC/UV/ICP-MS detection. Data collected on selenium proteins was evaluated in the context of individual animal trace element concentration, life history, and histological information. Selenium containing protein peak profiles varied in presence and intensity between animals with no pathological findings of cardiomyopathy and animals exhibiting evidence of cardiomyopathy. In particular, one class of proteins, metallothioneins, was found to be associated with Se and was in greater abundance in animals with cardiomyopathy than those with no pathological findings. Profiling Se species with SEC/ICP-MS proved to be a useful tool to identify Se protein pattern differences between heart disease stages in K. breviceps and an approach similar to this may be applied to other species to study Se protein associations with cardiomyopathy.

Electrochemical sensing of concanavalin A and ovalbumin interaction in solution

In an attempt to develop a label- and reagent-free electrochemical method for the detection of lectin-glycoprotein interactions, we tested lectin-concanavalin A (ConA), glycoprotein-ovalbumin (Ova) and their complex using chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode. Incubation of ConA with Ova resulted in an increase of the CPS peak H of the complex as compared to the CPS peaks of individual Ova and ConA proteins. Qualitatively similar results were obtained with other glycoprotein-lectin couples (ConA-RNase B and lectin from Sambucus nigra-fetuin). Using the CPS method, we were able to follow the course of complex formation in solution. Comparable responses of Ova, ConA and ConA-Ova complex were obtained not only at the mercury electrode but also with solid amalgam electrodes, which are more suitable for parallel analysis. It can be anticipated that electrochemical methods, namely CPS, will find application in glycomics and proteomics.

Univariate and multi-variate comparisons of protein and carbohydrate molecular structural conformations and their associations with nutritive factors in typical by-products.

Little attention has been paid on the inherent molecular structural effects among agricultural by-products. In this study, soybean meal (SM), wheat bran (WB), corn distillers dried grains with soluble (DDGS), dry brewer's grain (DBG), wet brewer's grain (WBG), and apple pomace (AP), which are widely used in the animal industry were selected to explore protein and carbohydrate molecular structural conformations. All the protein peak heights (including α-helix and β-sheet) and areas were exhibited highest values in SM and lowest in AP. The SM had the highest peak area intensity of cellulosic compounds (CELC), while the remaining varieties showed the lowest absorbance level. The TSCHO (sum of structural carbohydrate (SCHO) and CELC area exhibited variations among the samples. Multivariate comparisons showed AP had no molecular structural association with other by-products within the protein amide region. Protein amides I, II and (I+II) areas, α-helix, β-sheet and area ratio of protein amide and (TSCHO + TCHO) had strong relationships with CP, NDF, ADF, ADL, SCP, starch, PC, CA, CC and TDN contents. Inherent molecular structures varied among the selected by-product types and they might be used as potential predictors of nutritive factors, especially for protein structural information. © 2016 Society of Chemical Industry.

Screening of Serum Markers in Primary Immune Thrombocytopenia

To analyze the serum protein fingerprints of immune thrombocytopenia (ITP) patients and healthy controls by using weak cation exchange nanometer magnetic beads and MALDI-TOF-MAS technology, to identify the proteins with different expression, to establish the diagnostic model for ITP and to explore the pathogenesis of ITP. A total of 40 patients with ITP and 40 healthy controls were selected, the serum protein components were captured by using weak cation exchange nanaometer magnetic beads, the protein spectra of all specimens were detected by Autoflex II matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI- TOF-MS) and then the data were analyzed by CliprotoolsTM2.2 software, by which the distinct protein molecules were screened to set up ITP diagnostic model. To identify the established model, the sera of 20 ITP patients and 20 healthy controls were selected to make category and cross validations. The detection of Clinprot system and the analysis of CliprotoolsTM2.2 software showed that about 55 protein peaks were detected with the range of 700 Da to 10 000 Da of molecular weight in the protein spectrum of serum speciments from 40 ITP patients and 40 healthy controls. Compared with healthy controls, 19 protein expression peaks with statistically significant difference were found in ITP patients (P < 0.05), among them 5 expressions were up-regulated and 14 expressions were down-regulated. The diagnostic model on basis of Supervised Neural Network Algorithm (SNN) was established through 10 MS peaks with strongest capability in ITP group and control group automatically distinguished by software, and it is expected that the sensitivity of model group reached to 100%, and the specificity to 100%. The category validation showed that this diagnostic model correctly identificed all 20 ITP patients and 20 healthy controls, and in cross validation, the model sensitivity was 100% and the specificity was 100%. The ITP SNN model ertablished by using ChinProt System with high flax and good repetition is composed of 10 protein peaks with significant difference, this model can effectively distinguish ITP patients and healthy controls.

Molecular-Level Characterization of Normal, Benign, and Malignant Breast Tissues Using FTIR Spectroscopy

The present study investigates the diagnostic capability (sensitivity, specificity, and diagnostic accuracy) of the Fourier transform-infrared (FTIR) technique as a tool for the characterization of excised breast tissues with special emphasis on the differentiation between malignant and benign tumors. For such purpose, normal, malignant, and benign excised breast tissue samples were collected. The scatter diagrams of suggested FTIR characterization parameters were plotted and the corresponding diagnostic indices were calculated, showing maximum values reaching 100 % for differentiating between normal and diseased samples. The receiver operating characteristic analysis of such indices reported that the maximum area under the curve reached a value of one, which indicates highly accurate performance. The capability of the technique to differentiate between benign and malignant samples yielded promising results. A deconvolution of the Amide I protein peak revealed a high percentage of ordered protein secondary structure elements in normal tissue compared to those for malignant and benign tumors. The results indicate that the FTIR technique is ready for application in the characterization of excised breast tissues.