Food Web Implications of δ13C and δ15N Variability over 370 km of the Regulated Colorado River USA

Abstract

Dual stable isotope analysis in the regulated Colorado River through Grand Canyon National Park, USA, revealed a food web that varied spatially through this arid biome. Down-river enrichment of δ¹³C data was detected across three trophic levels resulting in shifted food webs. Humpack chub δ¹³C and δ¹⁵N values from muscle plugs and fin clips did not differ significantly. Humpback chub and rainbow trout trophic position is positively correlated with standard length indicating an increase in piscivory by larger fishes. Recovery of the aquatic community from impoundment by Glen Canyon Dam and collecting refinements for stable isotope analysis within large rivers are discussed.     

Simplified method for concentration of mitochondrial membrane protein complexes

Extracting and concentrating mitochondrial protein complexes from gel strips after blue native PAGE (BN‐PAGE) can be daunting tasks using the traditional methods, such as electroelution, passive diffusion and centrifugal concentration. We present a simplified gel electrophoresis method to concentrate mitochondrial protein complexes with excellent recovery rate. Mitochondrial complex I present in a long gel strip from BN‐PAGE can be easily concentrated into a 0.8 cm gel strip when a second BN‐PAGE is performed with a Y‐shaped gel and the addition of 0.01% n‐dodecyl β‐D ‐maltoside and 0.001% SDS in the cathode buffer. Once completed, the concentrated protein complex in the gel strip is ready for SDS‐PAGE or proteomic studies.     

Towards the total chemical synthesis of integral membrane proteins: a general method for the synthesis of hydrophobic peptide-thioester building blocks

Modification of a peptide-^αthioester with a sequence of six arginines on the thioester leaving group can render soluble all peptides derived from a polytopic integral membrane protein. This strategy greatly simplifies the synthesis of peptide-^αthioester building blocks for the total chemical synthesis of integral membrane proteins by native chemical ligation.     

High‐Mass MALDI‐MS Analysis for the Investigation of Protein Encapsulation within an Engineered Capsid Forming Protein

Chemical cross‐linking combined with MALDI ‐MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (AaLS ‐13) was investigated that self‐assembles into a capsid‐like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI ‐MS and native ESI ‐MS , were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo‐electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross‐linking with glutaraldehyde followed by MALDI ‐MS . The same approach was used to study protein‐protein interactions during encapsulation of GFP (+36) by the AaLS ‐13 capsid. Heterocomplexes between GFP (+36) and AaLS ‐13 multimers were not observed when mixed at maximal loading capacity (AalS‐13 monomer:GFP (+36) 4:1). This is in agreement with the known fast encapsulation of GFP (+36) by the protein capsid, which essentially removes any free GFP (+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP (+36) results in aggregation.     

Synthesis of a secretoglobin 3A2 type C (98–139) fragment by Dawson’s native chemical ligation

A secretoglobin 3A2 type C (98–139) peptide was synthesized by native chemical ligation between ¹¹⁵Ile and ¹¹⁶Cys residues using Dawson’s linker. The peptide-N-acyl-benzimidazolinone-glycine amide, a C-terminal thioesters precursor, was provided from 3-amino-4-(methylamino)benzoic acid. In addition, an N-terminal cysteine fragment, the (116–139) peptide, was prepared by ordinary Fmoc-solid phase peptide synthesis. Native chemical ligation of the (98–115) fragment with the Dawson’s linker and the (116–139) peptide smoothly proceeded to give SCGB3A2 type C (98–139) peptide.     

Sequential native chemical ligation utilizing peptide thioacids derived from newly developed Fmoc-based synthetic method

The first facile Fmoc-based synthetic procedure for peptide thioacids was developed. Successful application of the resulting thioacids to sequential native chemical ligation (NCL) in the N to C direction was achieved. Conversion of the peptide thioacids to the corresponding thioesters with Ellman's reagent followed by NCL in the presence of tris(2-carboxyethyl)phosphine (TCEP) and thiophenol was accomplished in a one-pot manner.     

Comparison of protein precipitation methods for various rat brain structures prior to proteomic analysis

Sample preparation is a fundamental step in proteomic methodologies. The quality of the results from a proteomic experiment is dependent on the nature of the sample and the properties of the proteins. In this study, various pre-treatment methods were compared by proteomic analysis; we analysed various rat brain structures after chloroform/methanol, acetone, TCA/acetone and TCA protein precipitation procedures. The protein content of the supernatant was also examined by 2-DE. We found that for four of the rat brain structures, precipitation with chloroform/methanol and acetone delivered the highest protein recovery for top-down proteomic analysis; however, TCA precipitation resulted in good protein separation and the highest number of protein spots in 2-DE. Moreover, TCA precipitation also gave high efficiency of protein recovery if prior sonication procedure was performed.     

Werkzeug für die Chemische Biologie. Semisynthese

Semisynthetic techniques have greatly contributed to the rapid development of Chemical Biology in recent years. In this regard the semisynthesis of complex modified proteins as well as the selective derivatization of natural products has evolved into more than mere proof‐of‐principle concepts but powerful tools to probe protein functions. This technology provides a solid basis for further investigations on proteomics and qualitative and quantitative cell biology. The interdisciplinary charter bridging chemistry and biology is the hallmark of semisynthesis. It can be expected that its scientific impact will further increase in the future.