Metabolic Fingerprinting of acs7 Mutant and Wild-Type Arabidopsis thaliana Under Salt Stress by Ultra Performance Liquid Chromatography Coupled with Quadrupole/Time of Flight Mass Spectrometry

The metabolic fingerprints of the acs7 mutant and wild-type (WT) of the model plant Arabidopsis thaliana with and without salt stress were compared by ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-QTOF MS). Separations were performed on C₁₈ column (2.1 mm × 100 mm, 1.7 µm). A linear gradient elution of acetonitrile and 0.1% formic acid solution was used at a flow rate of 0.4 mL/min. Principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were combined for the data treatment. A clear discrimination was obtained by both PCA and PLS-DA. The acs7 salt-treated group was closer to the control group samples than the WT salt-treated group samples. Several potential stress-induced ions were revealed as markers of salt stress. The markers 12-oxophytodienoic acid (OPDA), arabidopside A, sinapoyl malate, linolenic acid, and abscisic acid were identified by the accurate mass (from TOF MS). Linolenic acid and OPDA are the biosynthetic precursors of jasmonic acid (JA) by the octadecanoid pathway. The JA content determination results indicated that salt stress increased the JA levels in the leaves of the WT plant, but there was no significant increase in the JA content of acs7 after salt treatment. These data suggested the responses to salt stress of the acs7 mutant and WT A. thaliana were different in the octadecanoid pathway.     

Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey

Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC₅₀ values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents.     

High-resolution time-of-flight mass spectrometry fingerprinting of metabolites from cecum and distal colon contents of rats fed resistant starch

Time-of-flight mass spectrometry along with statistical analysis was utilized to study metabolic profiles among rats fed resistant starch (RS) diets. Fischer 344 rats were fed four starch diets consisting of 55 % (w/w, dbs) starch. A control starch diet consisting of corn starch was compared against three RS diets. The RS diets were high-amylose corn starch (HA7), HA7 chemically modified with octenyl succinic anhydride, and stearic-acid-complexed HA7 starch. A subgroup received antibiotic treatment to determine if perturbations in the gut microbiome were long lasting. A second subgroup was treated with azoxymethane (AOM), a carcinogen. At the end of the 8-week study, cecal and distal colon content samples were collected from the sacrificed rats. Metabolites were extracted from cecal and distal colon samples into acetonitrile. The extracts were then analyzed on an accurate-mass time-of-flight mass spectrometer to obtain their metabolic profile. The data were analyzed using partial least-squares discriminant analysis (PLS-DA). The PLS-DA analysis utilized a training set and verification set to classify samples within diet and treatment groups. PLS-DA could reliably differentiate the diet treatments for both cecal and distal colon samples. The PLS-DA analyses of the antibiotic and no antibiotic-treated subgroups were well classified for cecal samples and modestly separated for distal colon samples. PLS-DA analysis had limited success separating distal colon samples for rats given AOM from those not treated; the cecal samples from AOM had very poor classification. Mass spectrometry profiling coupled with PLS-DA can readily classify metabolite differences among rats given RS diets.     

多壁碳纳米管对质谱分析中的血清蛋白富集作用研究

通过多壁碳纳米管(MWCNTs)对临床血清蛋白提取物进行富集处理,经表面增强激光解析离子化飞行时间质谱(SELDI-TOF-MS)检测,发现MWCNTs对血清中小分子量蛋白(<20 kDa)具有很好的富集效果。同时还考察了内径、长度等参数对血清蛋白富集效果的影响。该方法可用于临床血清样本中低丰度的小分子量蛋白的检测。     

Affinity Matrices of Cratylia mollis Seed Lectins for Isolation of Glycoproteins from Complex Protein Mixtures

This work reports the use of matrices containing Cratylia mollis lectins (Cramoll 1,2,3-Sepharose and Cramoll 3-Sepharose) for isolation of glycoproteins from fetal bovine serum, human colostrum, hen egg white, and human blood plasma. Cramoll 1,2,3-Sepharose was able to bind a glycoprotein from fetal bovine serum which showed the same fetuin electrophoretic profile. The data indicate that this protein adsorbed to the matrix by interaction with Cramoll 3. Cramoll 1,2,3-Sepharose was not efficient to retain glycoproteins from human colostrum or commercial ovalbumin. Cramoll 3-Sepharose bound ovalbumin, and the support retained protein from hen egg white. Protein peaks eluted from the column with 1.0 M NaCl or 0.3 M galactose showed apparent molecular mass of ovalbumin. Two main proteins from blood plasma with apparent molecular mass 67 (similar to albumin) and 50 kDa (similar to fetuin) adsorbed on Cramoll 3-Sepharose and were eluted with 1.0 M NaCl as a single peak. Elution of adsorbed plasma proteins with 0.3 M galactose was less selective than with 1.0 M NaCl as revealed by SDS-PAGE. In conclusion, the Cramoll 1,2,3-Sepharose and Cramoll 3-Sepharose matrices were useful to separate glycoproteins from complex protein mixtures, and the adsorption phenomena was a carbohydrate-dependent event.     

磷酸盐洗脱液再生IMAC-Cu蛋白质芯片

使用扫描电子显微镜（SEM）和蛋白质芯片检测系统考察了磷酸盐洗脱液pH值和竞争洗脱剂对IMAC-Cu蛋白质芯片再生的影响。 结果表明,随着磷酸盐洗脱液pH值降低,芯片所吸附的蛋白质越容易被去除,但对芯片表面化学结构的破坏也随之加剧。 在洗脱液中加入竞争洗脱剂乙二胺四乙酸（EDTA）或甘氨酸（Gly）可有效改善蛋白质的洗脱效果。 当磷酸盐洗脱液pH=6.5、竞争洗脱剂为0.02 mol/L EDTA、洗脱时间72 h时,芯片的再生效果最佳。     

Multifactorial analysis of affinity-mass spectrometry data from serum protein samples: A strategy to distinguish patients with preeclampsia from matching control individuals

A multifactorial differential analysis of serum proteins using mass spectrometry distinguished samples from pregnant women with severe early-onset preeclampsia (n = 11) from those of control individuals with uneventful pregnancies (n = 13). Serum proteins were fractionated by either their affinities to reversed-phase material coated magnetic beads or by fractionated precipitation. The on-average most abundant ion signals were observed at m/z 9390, 9103, and 8886. The best differentiating ion signals between the two sample groups were found at m/z 13,715, 13,834, and 13,891. The normalized intensities of these ion signals were on-average lower in the preeclampsia group than in the control group. The six ion signal intensities enabled sorting of the individual spectra with high accuracy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a protein band migrating just above the 14 kDa marker band contained transthyretin (P02766; Mr (avg.): 13,761). Densitometric analysis of the transthyretin bands showed lower intensities in the preeclampsia samples with respect to those of the controls. Nephelometric analysis of the serum samples determined the mean concentration of transthyretin in the preeclampsia group were lower (0.16 mg/mL; range: 0.13 to 0.20; SD: 0.03) than that in the control group (0.19 mg/mL; range: 0.14 to 0.22; SD: 0.02), substantiating the role of transthyretin concentration differences in the comparison of the two groups. Altogether, our findings support the theory of preeclampsia being a heterogeneous disorder that might be sub-classified by a defined proteome signature in maternal blood using multifactorial analysis of affinity-fractionated serum samples.     

乙型肝炎病毒相关性肝癌患者的比较蛋白质组学研究

筛选并鉴定了乙型肝炎病毒(HBV)相关性肝癌的血清差异表达蛋白. 采用蛋白芯片及表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术对正常人与乙型肝炎病毒相关性肝癌患者术前血清分别进行检测, 共发现了44个差异蛋白, 其中21个上调, 23个下调. 通过高效液相色谱技术分离纯化其中表达差异最明显的蛋白, 并进行质谱鉴定. 通过蛋白功能结果分析表明, 这些蛋白的差异表达可能与乙型肝炎病毒相关性肝癌的发生机制密切相关.     

Classification of Rhizoma et Radix Notoperygii by HPLC Fingerprints with the Aid of Chemometrics

Chromatographic profiles of Rhizoma et Radix Notoperygii (RRN, “Qianghuo” in Chinese), a complex traditional Chinese medicine (TCM), were collected by high-performance liquid chromatography with diode array detection (HPLC-DAD) at 330 nm. These data profiles were used as fingerprints to investigate quality control classification modeling of the RRN samples. In contrast to the classical methods for discrimination of TCMs, that is, just using common HPLC peaks, all chromatographic profile data were pre-processed by the correlation optimized warping method and polynomial functions; then, these data were submitted as fingerprints (variables) for classification on the basis of sample origin. Chemometrics methods used for calibration modeling and subsequent sample classification-least square support vector machine (LS-SVM), artificial neural network (ANN), and partial least square discriminant analysis (PLS-DA); all produced satisfactory calibrations as well as classification results.     

Molybdenum Speciation in Raw Phloem Sap of Castor Bean

Abstract

Separation and quantification of molybdenum (Mo) in raw phloem sap from castor bean (Ricinus communis L.) was performed by size exclusion chromatography (SEC) and further purification was performed using quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE). For elemental detection, an inductively coupled plasma quadrupole mass spectrometer (ICP-QMS) was applied. Two different SEC columns were utilized: column A, Sephadex G-50 SF (700 mm × 24 mm), and column B, Sephadex G-25 M (28 mm × 9 mm). The protein content of the fractions was determined by the Bradford method. Using column A, two peaks of Mo were detected consisting of a main peak (Mo_A2) in the low molecular weight area (< 1.35 kDa), and a minor peak (Mo_A1, ≥ 30 kDa) at the void volume of the column. Both Mo species were detected at the ultraviolet (UV) active absorption area of raw phloem sap. Two peaks of Mo were also detected using column B, the first peak (Mo_B1) being at the same elution volume as the protein of raw phloem sap, and the second one (Mo_B2) was eluted in the area of 1.5 to 2.4 mL of elution volume. Raw phloem sap digested by proteinase K-enzyme indicates a significant reduction of Mo_B1 peak, which suggests that the peak may contain Mo bound to protein or polypeptides. The raw phloem sap and SEC fraction containing highest Mo concentration (Mo_A2) were furthermore separated by QPNC-PAGE. The result reveals that the Mo-containing fraction from the raw phloem sap was eluted at the same retention volume as the purified sample. This implies that the Mo species were also successfully separated and purified using QPNC-PAGE.     

Identification of differential signs of squamous cell lung carcinoma by means of the mass spectrometry profiling of blood plasma

The study deals with the search for potential biomarkers of squamous cell lung cancer by virtue of low-molecular weight proteome profiling (1–20 kDa) of blood plasma. The profiling was performed by automated sample preparation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The training set comprised of 152 blood plasma samples from patients with squamous cell lung cancer and 185 ones of the control group. For processing the profiles, the method of support vector machines was used in the environment of the ClinProTools 2.0 software and freely accessible language R for statistical analysis. The original software created in R-environment was shown to provide with more plentiful opportunities in the search for potential biomarkers, i.e., mass spectral peaks, in comparison to the ClinProTools 2.0 software. As a result of study, a number of 15 mass spectral peaks was established, which discriminated blood plasma samples collected from patients with squamous cell lung cancer and those of the control group. The selection of mass spectral peaks may potentially serve for the early diagnostics of the disease.     

Effects of flour or flaxseed oil upon testis mass in rats subjected to early weaning

Study evaluates testis mass in rats subjected to early weaning and subsequently nourished with diet containing flour or flaxseed oil. Pups were weaned for separation from mothers at 14 days (early weaning, EW) and 21 days (control, C). After 21 days, the control group (C60) was nourished with control diet. EW was divided as: control (EWC60), flaxseed flour (EWFF60) and flaxseed oil (EWFO60) group diets for the next 60 days. At 21st and 60th day, body mass, serum cholesterol and triglycerides and testis mass were evaluated. At 21 day, EW group showed lower (p < 0.05) body mass, serum cholesterol and testis mass. At 60 days, EWC60 and EWFO60 groups showed lower (p < 0.05) body mass (vs. C60 and EWFF60). EWFF60 group showed lower (p < 0.05) serum cholesterol (vs. EWC60 and EWFO60) and higher (p < 0.05) testis mass (vs. C60, EWC60 and EWFO60). Flaxseed flour (vs. oil) was associated with higher testis mass following early weaning.     

Profiling of hydroxyurea-treated β-thalassemia/ serum proteome through nano-LC–ESI–MS/ MS in combination with microsol-isoelectric focusing

Advancements in proteomic tools offer a comprehensive solution to studying the complexity of diseases at molecular level. This study focusses on the clinical proteomic profiling of pre- and post-hydroxyurea (HU)-treated β-thalassemia patients in parallel with healthy individuals to better understand the role of HU in the treatment of β-thalassemia. The strategy encompasses sequential high-resolution protein fractionation using MicroSol-isoelectric focusing (ZOOM- IEF) followed by one-dimensional SDS-PAGE before nano-RP-LC–MS/ MS analysis of tryptic peptides. Protein identification was performed through Mascot search using NCBInr and SwissProt databases. Several different proteins were observed in pool serum samples of each of the three study groups. Approximately, 1250 proteins exclusive to each group were identified, and after removing the redundant and low sequence coverage proteins, the number was reduced to 576 (201 in healthy, 187 in HU-untreated and 188 in HU-treated group). Uniquely identified proteins in the HU-treated group regulate the focal adhesion, ECM-receptor interaction, PI3K-Akt signaling, Rap1 signaling, cAMP signaling, platelet activation, and Ca²⁺ signaling pathways in the HU-treated group. The proteomic profile presented here will add to the current state of understanding of molecular mechanisms involved in hydroxyurea treatment of β-thalassemia.     

Metabolite Profiling to Monitor Organochlorine Pesticide Exposure in HepG2 Cell Culture

Gas chromatography coupled with time of flight mass spectrometry (GC/MS-TOF) was used to profile endogenous metabolites in HepG2 cell cultures to assess the metabolic changes induced by exposure to different organochlorine pesticides, their mixtures and controls (endosulfan, lindane, DDT and aldrin). Cells were cultured in DMEM with Glutamax at 37 °C with 5 % CO₂ for 72 h and then exposed to each pesticide, pesticide mixture or DMSO (as a control) for 24 h, and finally, endogenous metabolites were extracted and analyzed using GC/MS-TOF. The experiment was repeated six times under the same cell passage and culture conditions. PCA, PLS-DA and ROC were performed to analyze the GC/MS-TOF data and identify potential biomarkers. Thirty-five explanatory metabolites were found in both PCA and PLS-DA models, where Q ² was 0.86 and R ² was 0.98. Univariate and multivariate ROC showed potential biomarkers for each treatment, suggesting a general toxic mechanism for organochlorine pesticides that is specific for each type of compound. These results confirmed the effect of OCPs in sugar and amino acid metabolism that are linked with the function of cytochrome P450 in reductive dechlorination and oxidative stress.

     

Estimation of Protein Content in Plant Leaves using Spectral Reflectance: A Case Study in Euonymus japonica

Reflectance spectroscopy has been widely applied in the field of environmental studies. In this study, a low-cost, rapid, and nondestructive method using spectral reflectance was explored to evaluate protein concentrations in plant leaves of Euonymus japonica. Proteins in leaf samples were extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and five specific protein bands of interest were identified and quantified. Correlation analysis indicated that spectral reflectance had significant relationships with ribulose bisphosphate carboxylase a (r = ?0.43) and chlorophyll a-b binding protein (r = 0.53). A linear regression model and a quadratic regression model were formulated to directly and rapidly estimate the concentration of these two proteins with R ² = 0.61 and 0.7, respectively. To more accurately estimate the concentration of proteins, a precise inversion was established by a back propagation neutral network model using plant spectral absorption and position parameters, and the R ² values for proteins ribulose bisphosphate carboxylase, chlorophyll a-b binding protein, oxygen evolving enhancer protein, and ATP synthase subunit beta were 0.90, 0.91, 0.91, and 0.93, respectively. The models established in this study were shown to be useful tools for studies of plant biochemical components and health under different environmental conditions.     

MALDI-ToF mass spectrometry coupled with multivariate pattern recognition analysis for the rapid biomarker profiling of Escherichia coli in different growth phases

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) has been exploited extensively in the field of microbiology for the characterisation of bacterial species, the detection of biomarkers for early disease diagnosis and bacterial identification. Here, the multivariate data analysis technique of partial least squares-discriminant analysis (PLS-DA) was applied to ‘intact cell’ MALDI-ToF MS data obtained from Escherichia coli cell samples to determine if such an approach could be used to distinguish between, and characterise, different growth phases. PLS-DA is a technique that has the potential to extract systematic variation from large and noisy data sets by identifying a lower-dimensional subspace that contains latent information. The application of PLS-DA to the MALDI-ToF data obtained from cells at different stages of growth resulted in the successful classification of the samples according to the growth phase of the bacteria cultures. A further outcome of the analysis was that it was possible to identify the mass-to-charge (m/z) ratio peaks or ion signals that contributed to the classification of the samples. The Swiss-Prot/TrEMBL database and primary literature were then used to provisionally assign a small number of these m/z ion signals to proteins, and these tentative assignments revealed that the major contributors from the exponential phase were ribosomal proteins. Additional assignments were possible for the stationary phase and the decline phase cultures where the proteins identified were consistent with previously observed biological interpretation. In summary, the results show that MALDI-ToF MS, PLS-DA and a protein database search can be used in combination to discriminate between ‘intact cell’ E. coli cell samples in different growth phases and thus could potentially be used as a tool in process development in the bioprocessing industry to enhance cell growth and cell engineering strategies.     

Reproducibility of serum protein profiling by systematic assessment using solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

Protein profiling of human serum by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is potentially a new diagnostic tool for early detection of human diseases, including cancer. Sample preparation is a key issue in MALDI MS and the analysis of complex samples such as serum requires optimized, reproducible methods for handling and deposition of protein samples. Data acquisition in MALDI MS is also a critical issue, since heterogeneity of sample deposits leads to attenuation of ion signals in MALDI MS. In order to improve the robustness and reproducibility of MALDI MS for serum protein profiling we investigated a range of sample preparation techniques and developed a statistical method based on repeated analyses for evaluation of protein-profiling performance of MALDI MS. Two different solid-phase extraction (SPE) methods were investigated, namely custom-made microcolumns and commercially available magnetic beads. Using these two methods, nineteen different sample preparation methods for serum profiling by MALDI MS were systematically tested with regard to matrix selection, stationary phase, selectivity, and reproducibility. Microcolumns were tested with regard to chromatographic properties; reversed phase (C8, C18, SDB-XC), ion-exchange (anion, weak cation, mixed-phase (SDB-RPS)) and magnetic beads were tested with regard to chromatographic properties; reversed phase (C8) or affinity chromatography (Cu-IMAC). The reproducibility of each sample preparation method was determined by enumeration and analysis of protein signals that were detected in at least six out of nine spectra obtained by three triplicate analyses of one serum sample.A candidate for best overall performance as evaluated by the number of peaks generated and the reproducibility of mass spectra was found among the tested methods. Up to 418 reproducible peaks were detected in one cancer serum sample. These protein peaks can be part of a possible diagnostic profile, suggesting that this sample preparation method and data acquisition approach is suitable for large-scale analysis of serum samples for protein profiling.     

Purification and Characterization of Bowman‐Birk Protease Inhibitor from Rice Coleoptiles

A 15 kDa rice Bowman‐Birk inhibitor from fast elongating coleoptiles has been purified and identified using partial N‐terminal sequence, LC‐MS, and MALDI‐TOF MS as a 133 amino acid polypeptide (BBIrc 1). The kinetic study shows this protease inhibitor displays competitive inhibition toward trypsin with Ki of 4.0 × 10^?7 M and non‐competitive inhibition toward α‐chymotrypsin with Ki of 9.3 × 10^?6 M. The Western blotting results of the anti‐sera raised against this 15 kDa protein showed that this anti‐serum recognized two BBI proteins with molecular size around 15 kDa (BBIrc 1) and 25 kDa (BBIrc2) and the quantity of the expression of 15 kDa was nearly constant under both aerobic and hypoxia conditions; however, the 25 kDa expression was greatly up‐regulated when the fast elongating coleoptiles were transferred from hypoxia conditions to the aerobic conditions. The results indicate that the expression pattern of BBIs proteins correlated to the developmental stage in terms of morphological changes. The partial N‐terminal sequence of the first 9 amino acids of 25 kDa was AEAPPRPPK, which is the same as the amino acid sequence of 37th to 45th of RBBI3‐1 and LC‐MS study shows that several mass fragments fit to RBBI3‐1. The 25 kDa protein also shows specific binding to bovine trypsin. This expression pattern demonstrates for the first time that environmental factor, oxygen, can select and enhance specific BBI gene expression. The results of this study suggest BBI proteins might play multiple biological functions inside rice coleoptiles.     

Studies on the Toluidine Blue Dimer as a Fluorescence Probe for Protein Assays

Abstract

A spectrofluorometric method was developed for the determination of total serum protein by exploring toluidine blue (TB) as the fluorescence probe. The fluorescence intensity of TB at 648 nm was significantly quenched in the presence of sodium dodecylbenzene sulfonate (SDBS) by forming a dimer of the dye, which can afterwards reconvert to monomer when proteins were added accompanied by the recovery of the fluorescence. This might be attributed to the modulated transferring of the dimer‐monomer equilibrium of TB in the anionic surfactant caused by the addition of protein. A linear calibration graph was obtained in the range of 0.5–50 mg/l BSA, with a detection limit of 0.15 mg/l and a RSD of 1.3% (n=11, 5.0 mg/l BSA). Total proteins in human serums were analyzed by using the present procedure and the results agreed well with those obtained by the Biuret method.