Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol

IMAC can be used to selectively enrich phosphopeptides from complex peptide mixtures, but co-retention of acidic peptides together with the failure to retain some phosphopeptides restricts the general utility of the method. In this study Fe(III)-IMAC was qualitatively and quantitatively assessed using a panel of phosphopeptides, both synthetic and derived from proteolysis of known phosphoproteins, to identify the causes of success and failure in the application of this technique. Here we demonstrate that, as expected, peptides with a more acidic amino acid content are generally more efficiently purified and detected by MALDI-MS after Fe(III)-IMAC than those with a more basic content. Modulating the loading buffer used for Fe(III)-IMAC significantly affects phosphopeptide binding and suggests that conformational factors that lead to steric hindrance and reduced accessibility to the phosphate are important. The use of 1,1,1,3,3,3-hexa-fluoroisopropanol is shown here to significantly improve Fe(III)-IMAC enrichment and subsequent detection of phosphopeptides by MALDI-MS.

Liquid ultraviolet matrix-assisted laser desorption/ionization mass spectrometry for automated proteomic analysis

We have combined several key sample preparation steps for the use of a liquid matrix system to provide high analytical sensitivity in automated ultraviolet - matrix-assisted laser desorption/ ionisation - mass spectrometry (UV-MALDI-MS). This new sample preparation protocol employs a matrix-mixture which is based on the glycerol matrix-mixture described by Sze et al. U. Am. Soc. Mass Spectrom. 1998, 9, 166-174). The low-ferntomole sensitivity that is achievable with this new preparation protocol enables proteomic analysis of protein digests comparable to solid-state matrix systems. For automated data acquisition and analysis, the MALDI performance of this liquid matrix surpasses the conventional solid-state MALDI matrices. Besides the inherent general advantages of liquid samples for automated sample preparation and data acquisition the use of the presented liquid matrix significantly reduces the extent of unspecific ion signals in peptide mass fingerprints compared to typically used solid matrices, such as 2,5-dihydrox-ybenzoic acid (DHB) or alpha-cyano-hydroxycinnamic acid (CHCA). In particular, matrix and lowmass ion signals and ion signals resulting from cation adduct formation are dramatically reduced. Consequently, the confidence level of protein identification by peptide mass mapping of in-solution and in-gel digests is generally higher.

Serum proteomic abnormality predating screen detection of ovarian cancer

Ovarian cancer is characterized by vague, non-specific symptoms, advanced stage at diagnosis and poor overall survival. A nested case control study was undertaken on stored serial serum samples from women who developed ovarian cancer and healthy controls (matched for serum processing and storage conditions as well as attributes such as age) in a pilot randomized controlled trial of ovarian cancer screening. The unique feature of this study is that the women were screened for up to 7 years. The serum samples underwent prefractionation using a reversed-phase batch extraction protocol prior to MALDI-TOF MS data acquisition. Our exploratory analysis shows that combining a single MS peak with CA125 allows statistically significant discrimination at the 5% level between cases and controls up to 12 months in advance of the original diagnosis of ovarian cancer. Such combinations work much better than a single peak or CA125 alone. This paper demonstrates that mass spectra from the low molecular weight serum proteome carry information useful for early detection of ovarian cancer. The next step is to identify the specific biomarkers that make early detection possible.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye (TM) and analysed by two-dimensional difference gel. electrophoresis. Gel images analysed off-line, using the DeCyder (TM) image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

Formation of crystalline macrocyclic phases during electrophilic precipitation-polycondensation syntheses of poly(arylene ether ketone)s

Elongated crystalline particles formed as by-products during poly(arylene ether ketone) synthesis by electrophilic precipitation-polycondensation of 4,4'-diphenoxybenzophenone with terephthaloyl chloride or isophthaloyl chloride, thought previously to be polymer-whiskers, have now been identified as macrocyclic phases. Single crystal X-ray analysis of the needle-like particles formed in the reaction with terephthaloyl chloride, using the microdiffraction technique with synchrotron radiation, revealed that they consist of a macrocylic compound containing ten phenylene units, i.e. the [2 + 2] cyclic dimer. An analogous structure has also been demonstrated for the corresponding macrocycle derived from the reaction of 4,4-diphenoxybenzophenone with isophthaloyl chloride. Chloroform extraction of the products of the two polycondensations dissolved the macrocyclic material (but not the linear polymer), and analysis of the extracts by MALDI-TOF mass spectrometry demonstrated the presence in both cases of homologous families of macrocyclic products. Higher yields of macrocycles were obtained under pseudo-high dilution conditions, enabling the [2 + 2] cyclodimers from reactions of 4,4'-diphenoxybenzophenone with both terephthaloyl and isophthaloyl chloride to be isolated as pure compounds and fully characterised. (C) 2003 Published by Elsevier Ltd.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions (a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SIDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to similar to 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Possible evidence is presented for Maillard glycation of enzymes during oligosaccharide synthesis by reverse hydrolysis. In 70% (w/v) mannose solutions, 1,2-alpha-mannosidase from Penicillium citrinum lost 40% and alpha-mannosidase from almonds lost 60% activity at 55 degreesC over 2 weeks. Oligosaccharide yields were 15 and 45% respectively. Higher molecular weight glycation adducts were formed in a time-dependent manner as seen by MALDI-TOF. Inhibitors of the Maillard. reaction were able to partially alleviate these effects resulting in reduced loss of enzyme activity and oligosaccharide yield increases of 27-53% relative to the control. (C) 2004 Elsevier B.V. All rights reserved.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.

Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.)

Episodes of high temperature at anthesis, which in rice is the most sensitive stage to temperature, are expected to occur more frequently in future climates. The morphology of the reproductive organs and pollen number, and changes in anther protein expression, were studied in response to high temperature at anthesis in three rice (Oryza sativa L.) genotypes. Plants were exposed to 6 h of high (38 °C) and control (29 °C) temperature at anthesis and spikelets collected for morphological and proteomic analysis. Moroberekan was the most heat-sensitive genotype (18% spikelet fertility at 38 °C), while IR64 (48%) and N22 (71%) were moderately and highly heat tolerant, respectively. There were significant differences among the genotypes in anther length and width, apical and basal pore lengths, apical pore area, and stigma and pistil length. Temperature also affected some of these traits, increasing anther pore size and reducing stigma length. Nonetheless, variation in the number of pollen on the stigma could not be related to measured morphological traits. Variation in spikelet fertility was highly correlated (r=0.97, n=6) with the proportion of spikelets with ≥20 germinated pollen grains on the stigma. A 2D-gel electrophoresis showed 46 protein spots changing in abundance, of which 13 differentially expressed protein spots were analysed by MS/MALDI-TOF. A cold and a heat shock protein were found significantly up-regulated in N22, and this may have contributed to the greater heat tolerance of N22. The role of differentially expressed proteins and morphology during anther dehiscence and pollination in shaping heat tolerance and susceptibility is discussed.

Highly accurate detection of ovarian cancer using CA125 but limited improvement with serum matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling

Objectives: Our objective was to test the performance of CA125 in classifying serum samples from a cohort of malignant and benign ovarian cancers and age-matched healthy controls and to assess whether combining information from matrix-assisted laser desorption/ionization (MALDI) time-of-flight profiling could improve diagnostic performance. Materials and Methods: Serum samples from women with ovarian neoplasms and healthy volunteers were subjected to CA125 assay and MALDI time-of-flight mass spectrometry (MS) profiling. Models were built from training data sets using discriminatory MALDI MS peaks in combination with CA125 values and tested their ability to classify blinded test samples. These were compared with models using CA125 threshold levels from 193 patients with ovarian cancer, 290 with benign neoplasm, and 2236 postmenopausal healthy controls. Results: Using a CA125 cutoff of 30 U/mL, an overall sensitivity of 94.8% (96.6% specificity) was obtained when comparing malignancies versus healthy postmenopausal controls, whereas a cutoff of 65 U/mL provided a sensitivity of 83.9% (99.6% specificity). High classification accuracies were obtained for early-stage cancers (93.5% sensitivity). Reasons for high accuracies include recruitment bias, restriction to postmenopausal women, and inclusion of only primary invasive epithelial ovarian cancer cases. The combination of MS profiling information with CA125 did not significantly improve the specificity/accuracy compared with classifications on the basis of CA125 alone. Conclusions: We report unexpectedly good performance of serum CA125 using threshold classification in discriminating healthy controls and women with benign masses from those with invasive ovarian cancer. This highlights the dependence of diagnostic tests on the characteristics of the study population and the crucial need for authors to provide sufficient relevant details to allow comparison. Our study also shows that MS profiling information adds little to diagnostic accuracy. This finding is in contrast with other reports and shows the limitations of serum MS profiling for biomarker discovery and as a diagnostic tool

Evaluation of peak-picking algorithms for protein mass spectrometry

Peak picking is an early key step in MS data analysis. We compare three commonly used approaches to peak picking and discuss their merits by means of statistical analysis. Methods investigated encompass signal-to-noise ratio, continuous wavelet transform, and a correlation-based approach using a Gaussian template. Functionality of the three methods is illustrated and discussed in a practical context using a mass spectral data set created with MALDI-TOF technology. Sensitivity and specificity are investigated using a manually defined reference set of peaks. As an additional criterion, the robustness of the three methods is assessed by a perturbation analysis and illustrated using ROC curves.

Structural diversity of sainfoin (Onobrychis viciifolia) tannins

Tannins are oligomeric and polymeric polyphenols that are produced by many plants. The study of their biological activities is of interest because they can generate valuable nutritional, veterinary and environmental effects in ruminant livestock production [1]. Isolated tannin fractions from sainfoin (Onobrychis viciifolia), which is potentially a very useful animal feed, were characterised by MALDI-TOF MS and thiolytic degradation with benzyl mercaptan. Condensed tannins were analysed and characterised in more than different 40 sainfoin varieties to provide guidelines for future plant breeding programmes. Several different techniques were used to study these complex tannin mixtures.

Thiolytic screening method for exploring condensed tannin variation in a unique sainfoin germplasm bank

This EU funded 'HealthyHay'project stablished a sainfoin (Onobrychis vicifolia) germplasm bank at NIAB, Cambridge, with 306 accessions from around the world. A screening method was developed to characterise tannins by thiolytic degradation [1] directly in green plants for the first time. the method was validated by separate analysis of unextractable, extractable and purified tannins using thiolysis, HPLC-GPC and MALDI-TOF MS. Most tannins (58 to 73% of the total) could be recovered after Toyopearl HW50 fractionation with water, aqueous methanol and acetone. the greatest losses during purification occurred amongst larger molecular weight tannins with mean degree of polymerisation (mDP) > 18. The composition of water-,aqueous methanol- and acetone-soluble tannins differed considerably in their mDP and trans/cis ratios, but not in their prodelphinidin/orocyanidin (PD/PC) ratios.

Evaluating the effects of tannins on the extent and rate of in vitro measured gas and methane production using the Automated Pressure Evaluation System (APES)

An in vitro study was conducted to investigate the effect of tannins on the extent and rate of gas and methane production, using an automated pressure evaluation system (APES). In this study three condensed tannins (CT; quebracho, grape seed and green tea tannins) and four hydrolysable tannins (HT; tara, valonea, myrabolan and chestnut tannins) were evaluated, with lucerne as a control substrate. CT and HT were characterised by matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS). Tannins were added to the substrate at an effective concentration of 100 g/kg either with or without polyethylene glycol (PEG6000), and incubated for 72 h in pooled, buffered rumen liquid from four lactating dairy cows. After inoculation, fermentation bottles were immediately connected to the APES to measure total cumulative gas production (GP). During the incubation, 11 gas samples were collected from each bottle at 0, 1, 4, 7, 11, 15, 23, 30, 46, 52 and 72 h of incubation and analysed for methane. A modified Michaelis-Menten model was fitted to the methane concentration patterns and model estimates were used to calculate the total cumulative methane production (GPCH4). GP and GPCH4 curves were fitted using a modified monophasic Michaelis-Menten model. Addition of quebracho reduced GP (P=0.002), whilst the other tannins did not affect GP. Addition of PEG increased GP for quebracho (P=0.003), valonea (P=0.058) and grape seed tannins (P=0.071), suggesting that these tannins either inhibited or tended to inhibit fermentation. Addition of quebracho and grape seed tannins also reduced (P≤0.012) the maximum rate of gas production, indicating that microbial activity was affected. Quebracho, valonea, myrabolan and grape seed decreased (P≤0.003) GPCH4 and the maximum rate (0.001≤ P≤ 0.102) of CH4 production. Addition of chestnut, green tea and tara tannins did not affect total gas nor methane production. Valonea and myrabolan tannins have most promise for reducing methane production as they had only a minor impact on gas production.

Mass spectrometry imaging of glucosinolates in arabidopsis flowers and siliques

Glucosinolates are multi-functional plant secondary metabolites which play a vital role in plant defence and are, as dietary compounds, important to human health and livestock well-being. Knowledge of the tissue-specific regulation of their biosynthesis and accumulation is essential for plant breeding programs. Here, we report that in Arabidopsis thaliana, glucosinolates are accumulated differentially in specific cells of reproductive organs. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), distribution patterns of three selected compounds, 4-methylsulfinylbutyl (glucoraphanin), indol-3-ylmethyl (glucobrassicin), and 4-benzoyloxybutyl glucosinolates, were mapped in the tissues of whole flower buds, sepals and siliques. The results show that tissue localization patterns of aliphatic glucosinolate glucoraphanin and 4-benzoyloxybutyl glucosinolate were similar, but indole glucosinolate glucobrassicin had different localisation, indicating a possible difference in function. The high resolution images obtained by a complementary approach, cryo-SEM Energy Dispersive X-ray analysis (cryo-SEM-EDX), confirmed increased concentration of sulphur in areas with elevated amounts of glucosinolates, and allowed identifying the cell types implicated in accumulation of glucosinolates. High concentration of sulphur was found in S-cells adjacent to the phloem in pedicels and siliques, indicating the presence of glucosinolates. Moreover, both MALDI MSI and cryo-SEM-EDX analyses indicated accumulation of glucosinolates in cells on the outer surface of the sepals, suggesting that a layer of glucosinolate-accumulating epidermal cells protects the whole of the developing flower, in addition to the S-cells, which protect the phloem. This research demonstrates the high potential of MALDI MSI for understanding the cell-specific compartmentation of plant metabolites and its regulation.