Peptide barcoding for one-pot evaluation of sequence–function relationships of nanobodies

遊離型抗体の構造活性相関解析を迅速に評価可能とする新手法を開発. 京都大学プレスリリース. 2021-11-08.Optimisation of protein binders relies on laborious screening processes. Investigation of sequence–function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence–function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody–antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence–function relationships

Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans

単⼀神経細胞クラスのプロテオミクス解析を実現 --線⾍の神経細胞のプロテオームマップ構築に向けて--. 京都大学プレスリリース. 2020-08-17.Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function. Changes in protein synthesis are critical for neuronal function. Therefore, analysing protein expression patterns in individual neuronal subclass will elucidate molecular mechanisms for memory and other functions. In this study, we used neuronal subclass-selective proteomic analysis with cell-selective bio-orthogonal non-canonical amino acid tagging. We selected Caenorhabditis elegans as a model organism because it shows diverse neuronal functions and simple neural circuitry. We performed proteomic analysis of all neurons or AFD subclass neurons that regulate thermotaxis in C. elegans. Mutant phenylalanyl tRNA synthetase (MuPheRS) was selectively expressed in all neurons or AFD subclass neurons, and azido-phenylalanine was incorporated into proteins in cells of interest. Azide-labelled proteins were enriched and proteomic analysis was performed. We identified 4, 412 and 1, 834 proteins from strains producing MuPheRS in all neurons and AFD subclass neurons, respectively. F23B2.10 (RING-type domain-containing protein) was identified only in neuronal cell-enriched proteomic analysis. We expressed GFP under the control of the 5′ regulatory region of F23B2.10 and found GFP expression in neurons. We expect that more single-neuron specific proteomic data will clarify how protein composition and abundance affect characteristics of neuronal subclasses

A critical role of an oxygen-responsive gene for aerobic nitrogenase activity in Azotobacter vinelandii and its application to Escherichia coli

酸素に弱いニトロゲナーゼを有酸素条件でも機能させるための重要因子を発見 --化石燃料を必要としない脱炭素社会の基盤形成に向け、空中窒素の固定を介した生物学的アンモニア生産へ期待--. 京都大学プレスリリース. 2022-03-17.Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions

Definitive screening design enables optimization of LC–ESI–MS/MS parameters in proteomics

<p>In proteomics, more than 100,000 peptides are generated from the digestion of human cell lysates. Proteome samples have a broad dynamic range in protein abundance; therefore, it is critical to optimize various parameters of LC–ESI–MS/MS to comprehensively identify these peptides. However, there are many parameters for LC–ESI–MS/MS analysis. In this study, we applied definitive screening design to simultaneously optimize 14 parameters in the operation of monolithic capillary LC–ESI–MS/MS to increase the number of identified proteins and/or the average peak area of MS1. The simultaneous optimization enabled the determination of two-factor interactions between LC and MS. Finally, we found two parameter sets of monolithic capillary LC–ESI–MS/MS that increased the number of identified proteins by 8.1% or the average peak area of MS1 by 67%. The definitive screening design would be highly useful for high-throughput analysis of the best parameter set in LC–ESI–MS/MS systems.</p> <p>Definitive screening design enables simultaneous optimization of LC-ESI-MS/MS parameters in proteomics.</p

Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins.

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions

モノリス型カラムを用いた尿中メタンフェタミンの抽出

We have established a simple screening method for methamphetamine (MA) in urine using a monolithic silica capillary column for extraction and using the Simon's reagent for colorimetric determination. Urine was mixed with borate buffer solution (50 mM, pH 10.2) and the mixture was passed through the column connected with the needle of a gas-tight syringe. MA in urine was adsorbed to the column, then eluted with 10 µl of ethyl acetate, and directly spotted on a thin-layer chromatographic plate by dividing the eluate into several aliquots. To separate MA from urine impurities and detect it clearly, a 2-µl volume of a solvent mixture (chloroform : methanol = 95 : 5, v/v, saturated with 28-0x1.fa060bfff8bbp+0mmonium hydroxide) was applied to the spot for concentrically development of the analyte. After drying the solvent, 0.5% sodium nitroprusside solution containing 10% sodium carbonate was sprayed on the plate. The plate was put in a bottle saturated with acetaldehyde, and the ring was changed purple (Simon's reaction) when MA existed in urine. The macroscopic detection limit of MA in urine by this method was 0.5 µg/ml. To confirm efficiency of this method, the eluate from the column was analyzed by GC/MS using penta-deuterated MA as internal standard. By analysis of 30 urine samples, the results obtained by the present spot test agreed well with those by GC/MS