Solvent Mediated Assembly of Nanoparticles Confined in Mesoporous Alumina

The controlled self-assembly of thiol stabilized gold nanocrystals in a mediating solvent and confined within mesoporous alumina was probed in situ with small angle x-ray scattering. The evolution of the self-assembly process was controlled reversibly via regulated changes in the amount of solvent condensed from an undersaturated vapor. Analysis indicated that the nanoparticles self-assembled into cylindrical monolayers within the porous template. Nanoparticle nearest-neighbor separation within the monolayer increased and the ordering decreased with the controlled addition of solvent. The process was reversible with the removal of solvent. Isotropic clusters of nanoparticles were also observed to form temporarily during desorption of the liquid solvent and disappeared upon complete removal of liquid. Measurements of the absorption and desorption of the solvent showed strong hysteresis upon thermal cycling. In addition, the capillary filling transition for the solvent in the nanoparticle-doped pores was shifted to larger chemical potential, relative to the liquid/vapor coexistence, by a factor of 4 as compared to the expected value for the same system without nanoparticles.Comment: 9 pages, 9 figures, appeared in Phys. Rev.

Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC) and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥ 65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.status: publishe

Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry

The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization--tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT.status: publishe

Improved identification of wheat prolamins through alkylation of cysteine residues and peptide-based mass spectrometry

Wheat flour quality greatly depends on the composition of its prolamins and the presence and location of cysteine residues in most of them. Wheat prolamin detection and quantification is also relevant in the context of celiac disease, especially since the commonly used ELISA technique often produces inconsistent results. LC-MS/MS, which has greatly advanced in recent years, is very useful for protein quantification and structure identification, e.g. to analyze the ratio of thiol and disulfide groups as affected by processing. In addition, isotope coded affinity tags (ICAT) for cysteine residues are increasingly used for quantitative proteomics. However, in the case of wheat prolamins, these techniques suffer from the low solubility of prolamins in buffers typically used for sample preparation. Here, methods are presented for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM), which significantly improve the detection of cysteine-containing peptides that can then be identified in enzymic prolamin digests by ESI-MS/MS. Optimal conditions for cysteine labeling, enzymic digestion of wheat prolamins, and detection of cysteine peptides are discussed. Finally, to the best of our knowledge, we were the first to label prolamins with a commonly used ICAT reagent. However, the extent of labeling was much higher with 4-VP and IDAM than with ICAT. The new protocol including alkylation with IDAM or 4-VP and LC-MS/MS of wheat prolamins offers perspectives for elucidation of the gluten structure and determination of gluten concentrations in (gluten-free) products.status: publishe

Relative average molecular masses (M_r) and corresponding proteins detected in the isolated HMW-GS fractions of the cvs. Akteur, Contra and Apache as determined by RP-HPLC-ESI-MS with the mobile phase containing 0.1% (v/v) TFA.

1<p>Standard deviations are given between brackets.</p>2<p>Based on M_r agreement with proteins from the wheat storage proteome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Lagrain1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Dupont1" target="_blank">[36]</a>.</p

Mass spectra of wheat HMW-GS Ax1 and ω5-gliadin.

<p>The spectra are taken from the average of scans under the peak with retention time 6.2 min from the base peak mass chromatogram after RP-HPLC-ESI-MS with 0.1% (v/v) formic acid in the mobile phase. The simulated mass spectra obtained by maximum entropy deconvolution are shown as insets.</p

Base peak mass chromatogram from RP-HPLC-ESI-MS of the isolated HMW-GS fraction of wheat cv. Akteur.

<p>The mobile phase contains 0.1% (v/v) TFA. HMW-GS are labeled with numbers according to the current nomenclature system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Payne2" target="_blank">[4]</a>.</p

Relative average molecular masses (M_r) and corresponding proteins present in the isolated HMW-GS fractions of the cv. Akteur as determined by RP-HPLC-ESI-MS with the mobile phase containing 0.1% (v/v) formic acid.

1<p>Standard deviations are given between brackets.</p>2<p>Based on M_r agreement with proteins from the wheat storage proteome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Lagrain1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Mamone1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Muccilli1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058682#pone.0058682-Dupont1" target="_blank">[36]</a>.</p

Levels of ω5-gliadins and HMW-GS in flour and isolated HMW-GS fractions of the wheat cvs. Akteur, Contra and Apache and the respective protein yields in the HMW-GS fractions.

1<p>Including the glutenin-bound (ωb-) gliadins.</p