A proteomic analysis of organelles from Arabidopsis thaliana

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.     

高通量蛋白质组学分析研究进展

基于质谱的蛋白质组学技术已经日趋成熟,可以对细胞和组织中的成千上万种蛋白质进行全面的定性和定量分析,逐步实现“深度覆盖”。随着生物医学日益增长的大队列蛋白质组学分析需求,如何在保持较为理想的覆盖深度下实现短时间、快速的“高通量”蛋白质组学分析已成为当前亟需解决的关键问题之一。常规的蛋白质组学分析流程通常包括样品前处理、色谱分离、质谱检测和数据分析。该文从以上4个方面展开介绍近10年以来高通量蛋白质组学分析技术取得的一系列研究进展,主要包括:(1)基于高通量、自动化移液工作站的蛋白质组样品前处理方法;(2)基于微升流速液相色谱与质谱联用的高通量蛋白质组检测方法;(3)利用灵敏度高、扫描速度快的质谱仪实现短色谱梯度分离下蛋白质组深度覆盖的分析方法;(4)基于人工智能、深度神经网络、机器学习等的蛋白质组学大数据分析方法。此外,对高通量蛋白质组学面临的挑战及其发展进行展望。总而言之,预期在不久的将来高通量蛋白质组学技术将会逐步“落地转化”,成为大队列蛋白质组学分析的利器。     

Identification of novel vaccine candidates against carbapenem resistant Klebsiella pneumoniae: A systematic reverse proteomic approach

Klebsiella pneumoniae is declared as antibiotic resistant by WHO, with the critical urgency of developing novel antimicrobial therapeutics as drug resistance is the second most dangerous threat after terrorism. Besides many attempts still, there is no effective vaccine available against K. pneumoniae. By utilizing all the available proteomic data we prioritized the novel proteins ideal for vaccine development using bioinformatics tools and techniques. Among the huge data, eight proteins passed all the barriers and were considered ideal candidates for vaccine development. These include: copper silver efflux system outer membrane protein (CusC), outer membrane porin protein (OmpN), Fe⁺⁺ enterobactin transporter substrate binding protein (fepB), zinc transporter substrate binding protein (ZnuA), ribonuclease HI, tellurite resistant methyltransferase (the B), and two uncharacterized hypothetical proteins (WP_002918223 and WP_002892366). These proteins were also subjected to epitope analysis and were found best for developing subunit vaccine against K. pneumoniae. The study shows that the potential vaccine targets are sufficiently efficient being virulent, of outer membranous origin and can be proposed for the DNA third-generation vaccines development that would help to cope up infections caused by multidrug-resistant K. pneumoniae.     

A proteomic analysis of secreted proteins from xylan-induced Bacillus sp. strain K-1

The expression level of extracellular proteins in an alkaliphilic bacterium, Bacillus sp. strain K-1, grown in a xylan-containing medium, is significantly increased when compared with that grown in the nonxylan culture medium. A proteomic approach has been efficiently applied to separate and characterize these differentially expressed secretory proteins. Eight prominent protein spots were identified and subjected to N-terminal amino acid sequencing. The results show that three spots share considerable similarity with the xylanolytic enzymes and that two spots share considerable similarity with the GltC regulatory protein and 3-dehydroquinate dehydratase, respectively. In addition, the three other proteins show little similarity with the known proteins in the database. In conclusion, our results demonstrate that the proteomic approach is a highly efficient method to rapidly study the differential expression of the secreted proteins by Bacillus sp. strain K-1 grown under xylan-induced condition.     

Aromatic Esters of Bicyclic Amines as Antimicrobials against Streptococcus pneumoniae

A double approach was followed in the search of novel inhibitors of the surface choline‐binding proteins (CBPs) of Streptococcus pneumoniae (pneumococcus) with antimicrobial properties. First, a library of 49 rationally‐designed esters of alkyl amines was screened for their specific binding to CBPs. The best binders, being esters of bicyclic amines (EBAs), were then tested for their in vitro effect on pneumococcal growth and morphology. Second, the efficiency of EBA‐induced CBP inhibition was enhanced about 45 000‐fold by multivalency effects upon synthesizing a poly(propylene imine) dendrimer containing eight copies of an atropine derivative. Both approaches led to compounds that arrest bacterial growth, dramatically decrease cell viability, and exhibit a protection effect in animal disease models, demonstrating that the pneumococcal CBPs are adequate targets for the discovery of novel antimicrobials that overcome the currently increasing antimicrobial resistance issues.     

Quantitative proteomic analysis of chromatin-associated factors

A method to identify and quantify chromatin-associated proteins has been developed and applied to the analysis of changes in chromatin-associated proteins induced by Myc oncoprotein expression in human B lymphocytes. Chromatin-enriched fractions were isolated by differential detergent/salt extraction and analyzed by ICAT reagent labeling, multi-dimensional chromatography and tandem mass spectrometry. Many known chromatin-associated regulatory factors were identified and quantified. The method will be widely applicable to various biological systems and reveal changes in chromatin-associated regulatory factors that underlie biological phenomena.     

蛋白质甲基化修饰的蛋白质组学分析方法新进展

蛋白质的甲基化修饰是一类重要的翻译后修饰。但与磷酸化、糖基化和泛素化等翻译后修饰相比,甲基化修饰的蛋白质组学分析方法开发还是一个较新的研究领域。近几年,由于甲基化修饰在表观遗传调控中的重要作用,这一修饰类型得到了越来越多的关注,相关的分析技术和分析方法也取得了较多进展。其中,基于质谱的蛋白质组学分析方法在甲基化修饰中发挥着关键的作用,实现了这一甲基化修饰的高通量分析。该综述将从甲基化修饰的分离富集、假阳性率控制以及定量蛋白质组学等方面对一些蛋白质甲基化修饰的分析技术和方法的最新进展进行介绍。     

Block Synthesis of Streptococcus pneumoniae Type 14 Capsular Polysaccharide Structures*

Synthesis of a thioglycoside tetrasaccharide block, β‐D‐Galp‐(1→4)‐β‐DGlcp‐(1→6)‐[β‐D‐Galp‐(1→4)]‐β‐D‐GlcNPhthp‐(1→SEt, corresponding to the repeating unit of Streptococcus pneumoniae serotype 14 CPS is described. Coupling of this block with a spacer followed by removal of an isopropylidene acetal yielded an acceptor, which was elongated with the donor block to give a protected dimer of the repeating unit. Iteration of this methodology yielded the trimer. Deprotection then produced an octa and a dodecasaccharide derivative ready for conjugation to proteins to afford immunoactive glycoconjugates.     

Synthetic directions towards capsular polysaccharide of Streptococcus pneumoniae serotype 18C

An efficient synthetic strategy has been developed for the synthesis of the tri, tetrasaccharide block and pentasaccharide corresponding to the capsular polysaccharide of Streptococcus pneumonia serotype 18C as their 2-aminoethyl glycosides. A one-pot glycosylation-deprotection, sequential glycosylations strategy has been adopted for the construction of the fragments and pentasaccharide derivative, which were then transformed into target compound after a series of functional group transformations. The synthetic method relies on the use of p-methoxybenzyl ether as an in situ-removable protecting group to reduce the number of reaction steps significantly. Here H₂SO₄-silica has been used successfully as a promoter for all glycosylation reaction. In addition, the synthetic target also contained a free amino group at its reducing end, facilitating its conjugation with other molecules for various biological studies and applications.     

Transition-state analysis of S. pneumoniae 5'-methylthioadenosine nucleosidase

Kinetic isotope effects (KIEs) and computer modeling are used to approximate the transition state of S. pneumoniae 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN). Experimental KIEs were measured and corrected for a small forward commitment factor. Intrinsic KIEs were obtained for [1'-3H], [1'-14C], [2'-3H], [4'-3H], [5'-3H(2)], [9-15N] and [Me-3H(3)] MTAs. The intrinsic KIEs suggest an SN1 transition state with no covalent participation of the adenine or the water nucleophile. The transition state was modeled as a stable ribooxacarbenium ion intermediate and was constrained to fit the intrinsic KIEs. The isotope effects predicted a 3-endo conformation for the ribosyl oxacarbenium-ion corresponding to H1'-C1'-C2'-H2' dihedral angle of 70 degrees. Ab initio Hartree-Fock and DFT calculations were performed to study the effect of polarization of ribosyl hydroxyls, torsional angles, and the effect of base orientation on isotope effects. Calculations suggest that the 4'-3H KIE arises from hyperconjugation between the lonepair (n(p)) of O4' and the sigma* (C4'-H4') antibonding orbital owing to polarization of the 3'-hydroxyl by Glu174. A [methyl-3H(3)] KIE is due to hyperconjugation between np of sulfur and sigma* of methyl C-H bonds. The van der Waal contacts increase the 1'-3H KIE because of induced dipole-dipole interactions. The 1'-3H KIE is also influenced by the torsion angles of adjacent atoms and by polarization of the 2'-hydroxyl. Changing the virtual solvent (dielectric constant) does not influence the isotope effects. Unlike most N-ribosyltransferases, N7 of the leaving group adenine is not protonated at the transition state of S. pneumoniae MTAN. This feature differentiates the S. pneumoniae and E. coli transition states and explains the 10(3)-fold decrease in the catalytic efficiency of S. pneumoniae MTAN relative to that from E. coli.     

Mechanism for the degradation of erythromycin A and erythromycin A 2'-ethyl succinate in acidic aqueous solution

A major drawback of the antibiotic erythromycin A is its extreme acid sensitivity, leading to rapid inactivation in the stomach. The accepted model for degradation in aqueous acidic solution has erythromycin A in equilibrium with erythromycin A enol ether and degrading to anhydroerythromycin A. We report a detailed kinetic study of the acidic degradation of erythromycin A and of erythromycin A 2'-ethyl succinate (the market-leading pediatric prodrug), investigating the reaction rates and degradation products via NMR. This reveals that the accepted mechanism is incorrect and that both the enol ether and the anhydride are in equilibrium with the parent erythromycin. By implication, both the anhydride and enol ether are antibacterially inactive reservoirs for the parent erythromycin. The actual degradation pathway is the slow loss of cladinose from erythromycin A (or erythromycin A 2'-ethyl succinate), which is reported here for the first time in a kinetic study. The kinetic analysis is based on global, nonlinear, simultaneous least-squares fitting of time course concentrations for all species across multiple datasets to integrated rate expressions, to provide robust estimates of the rate constants.     

Synthesis of the core structure of the lipoteichoic acid of Streptococcus pneumoniae

Streptococcus pneumoniae LTA is a highly complex glycophospholipid that consists of nine carbohydrate residues: three glucose, two galactosamine and two 2‐acetamino‐4‐amino‐2,4,6‐trideoxygalactose (AATDgal) residues that are each differently linked, one ribitol and one diacylated glycerol (DAG) residue. Suitable building blocks for the glucose and the AATDgal residues were designed and their synthesis is described in this paper. These building blocks permitted the successful synthesis of the core structure Glcβ(1‐3)AATDgalβ(1‐3)Glcα(1‐O)DAG in a suitably protected form for further chain extension ( 1 b , 1 c ) and as unprotected glycolipid ( 1 a ) that was employed in biological studies. These studies revealed that 1 a as well as 1 lead to interleukin‐8 release, however not via TLR2 or TLR4 as receptor.     

Photopolymerized microtips for sample preparation in proteomic analysis

We demonstrate a novel method for the fabrication of disposable plastic microtips, which we name "EasyTip", by a photopolymerization technique. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The fabricated EasyTips can be manipulated using commercial pipettes for wash/elution of minute amount of biological samples (< 10 microL) and can be applied for mass spectrometry (MS)-based proteomic analysis, in which the detection sensitivity depends critically on the optimal sample preparation. The recovery of a sample of 25 fmol of tryptic hemoglobin digest loaded in a C18 EasyTip was near 100% and we estimated the loading capacity to be around 0.4-2.0 microg of total proteins or peptides, which is well above a sufficient quantity for MS analysis. The effectiveness of the C18 EasyTips in enhancing the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MS signal, and thus providing a greater sequence coverage, was also demonstrated by the analysis of hemoglobin digest and the in-gel digested epidermal growth factor receptor (EGFR) protein from A431 cell lysate. We also demonstrated the usefulness of the immobilized IMAC EasyTips in extracting the signal of tryptic phosphopeptides of beta-casein (10 pmol) having one and four phosphorylation sites by using an IMAC EasyTip prior to off-line analysis by MS. The combination of IMAC EasyTips and MALDI-MS allowed the unambiguous identification of phosphopeptides based on the phosphatase assay as well as the post-source decay. Compared to other miniaturized devices, this fabrication method is simple, cheap, and requires less human intervention. Moreover, the method of manipulating the EasyTips is straightforward and can be automated readily by a robotic system for high-throughput analysis.     

Synthesis of the repeating unit of the lipoteichoic acid of Streptococcus pneumoniae

The lipoteichoic acid repeating unit of Streptococcus pneumoniae is a complex pseudopentasaccharide (3). It consists of one ribitol-phosphate, one 2-acetamino-4-amino-2,4,6-trideoxy-galactose, one glucose and two galactosamine residues each differently linked, but both carrying one phosphocholine substituent, at position 6. Suitable building blocks (6–10) for efficient and diastereocontrolled ligations were designed, thus providing, after complete deprotection, the target molecule in high purity. Biological tests revealed that repeating unit 3, lacking the lipid moiety, did not stimulate a pro-inflammatory response in human monocytes (hMNCs).     

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.     

Novel erythromycin A 9-tritylhydrazone: selective 6-O-methylation and conformational analysis

The straightforward synthesis of the novel 9-tritylhydrazone erythromycin A and its highly regioselective O-methylation at C(6)-OH are described. Preliminary conformational data based on X-ray diffraction, ¹H NMR and molecular mechanics is also presented with the aim of understanding the observed high regioselectivity. The facile synthesis of 6,12-O-dimethylerythromycin A, a standard to assess clarithromycin purity in quality control processes, is reported as well.     

Recent stationary phase-based fractionation strategies in proteomic analysis

In this article, we review stationary phase-assisted sample prefractionation in proteomic analysis in 2019–2022 period. Applications are grouped according to the mode of retention used in the prefractionation step prior to the final analysis by low-pH reversed-phase liquid chromatography-tandem mass spectrometry. Online or offline instrumental configurations are also commented on, with a highlight on novel online platforms. From the articles reviewed in this period, the order of popularity of chromatographic modes for sample fractionation is affinity chromatography > size exclusion chromatography > hydrophilic interaction chromatography > high-pH reversed-phase liquid chromatography > ion exchange chromatography.     

Shotgun proteomic analysis of Arabidopsis thaliana leaves

Two shotgun tandem MS proteomics approaches, multidimensional protein identification technology (MudPIT) and 1-D gel-LC-MS/MS, were used to identify Arabidopsis thaliana leaf proteins. These methods utilize different protein/peptide separation strategies. Detergents not compatible with MudPIT were used with 1-D gel-LC-MS/MS to help enrich for the detection of membrane-spanning and hydrophobic proteins. By combining the data from all MudPIT and 1-D gel-LC-MS/MS experiments, 2342 nonredundant proteins spanning a broad range of molecular weights and pI values were detected. With the exception of unknown proteins, the distribution of gene ontology (GO) classifications for the detected proteins was similar to that encoded by the genome, which shows that these extraction and separation procedures are useful for a broad proteomic survey of plant cells. Unknown proteins will likely have to be targeted by using additional methods, some of which should be compatible with separation strategies taken here.