Identification of phosphatidic acid interacting proteins in Ganoderma lingzhi / 生物工程学报

Ganoderma lingzhi is widely recognized as a medicinal basidiomycetes. Triterpene acids (TAs) are the key bioactive medicinal components of G. lingzhi. Our previous studies have shown that phospholipid acid (PA) produced by phospholipase D (PLD) plays a regulatory role in TA synthesis. In order to further elucidate the molecular mechanism how PA regulates TA synthesis in G. lingzhi, PA beads enrichment combined with LC-MS/MS technology was used to identify PA interacting proteins in G. lingzhi. A total of 19 PA interacting proteins were identified, including cytochrome P450 monooxygenase (GL22084), specific protein kinase MAPK (GL23765), catalase and cell surface hydrophobicity-associated protein. GST tagged GL22084 and GL23765 proteins were obtained through gene cloning, heterologous expression, and purification. The interactions between GL22084/GL23765 and PA were verified by GST pull down assay. The identification of PA interacting proteins provides a basis for further understanding the molecular mechanism how PLD-mediated PA signaling molecules regulates the TA synthesis in G. lingzhi. Moreover, the PA interacting proteins identified in this study can also provide clues for the research of PLD/PA signaling pathway in other species.

Protein fingerprinting with digital sequences of linear protein subsequence volumes: a computational study

Proteins in a proteome can be identified from a sequence of K integers equal to the digitized volumes of subsequences withL residues from the primary sequence of a stretched protein. Exhaustive computations on the proteins of Helicobacterpylori (UniProt id UP000000210) with L and K in the range 4–8 show that *90% of the proteins can be identified uniquelyin this manner. This computational result can be translated into practice with a nanopore, an emerging technology that doesnot require analyte immobilization, proteolysis or labeling. Unlike other methods, most of which focus on a specific targetprotein, nanopore-based methods enable the identification of multiple proteins from a sample in a single run. Recent workby Kennedy, Kolmogorov and associates shows that the blockade current due to a protein molecule translocating through ananopore is roughly proportional to one or more contiguous residues. The present study points to a modified version inwhich the volumes of subsequences (rather than of single residues) may be obtained by integrating the blockade current dueto L contiguous residues. The advantages arising from this include lower detector bandwidth, elimination of thehomopolymer problem and reduced noise. Because an identifier is based on near as well as distant (up to 2KL-L) residues,this approach uses more global information than an approach based on single residues and short-range correlations. Theresults of the study, which are available in a data supplement, are discussed in detail. Potential implementation issues areaddressed.

Morphological Trait, Molecular Genetic Evidence and Proteomic Determination of Different Chickens (Gallus gallus) Breeds

The phenotypic characterization, genetic variation and proteomic analysis of three main male chicken (Gallus gallus) breeds were investigated. These included hybrid red jungle fowl with the native chicken breed (KaiTor), white tail yellow chicken (WTYC) and commercial layer hen (HL). A phenetic analysis found that two major clades were observed in which the first two clades of Kai-Tor (clade A) and HL (clade B) were related. Meanwhile, WTYC was distinctly separated from the others. In terms of genetic diversity, three haplotypes were observed with 0.343 ± 0.097 of haplotypes diversity (Hd). The nucleotide diversity (Pi) of all samples was 0.00057 which conformed to low genetic diversity. In terms of protein characterization, two potential protein biomarkers were found in Kai-Tor serum samples namely 1) ATP-dependent RNA helicase DHX34 (DHX34; Accession number: XP_015128539.1) and 2) histone-lysine N-methyltransferase SETDB1 isoform X6 (SETDB1; Accession number: XP_015135538.1). Only one biomarker peptide was detected in HL (Cell division cycle 7-related protein kinase; CDC7; Accession number: XP_422347.5) as well as in WTYC (Bloom syndrome protein; BLM; Accession number: Q9I920).

Production optimization, purification, expression, and characterization of a novel α-L-arabinofuranosidase from Paenibacillus polymyxa

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.

Progress in the spectral library based protein identification strategy / 生物工程学报

Exponential growth of the mass spectrometry (MS) data is exhibited when the mass spectrometry-based proteomics has been developing rapidly. It is a great challenge to develop some quick, accurate and repeatable methods to identify peptides and proteins. Nowadays, the spectral library searching has become a mature strategy for tandem mass spectra based proteins identification in proteomics, which searches the experiment spectra against a collection of confidently identified MS/MS spectra that have been observed previously, and fully utilizes the abundance in the spectrum, peaks from non-canonical fragment ions, and other features. This review provides an overview of the implement of spectral library search strategy, and two key steps, spectral library construction and spectral library searching comprehensively, and discusses the progress and challenge of the library search strategy.

Fragmentation Characteristics and Utility of Immonium Ions for Peptide Identification by MALDI TOF/TOF Spectrometry / 分析化学

One of significant characteristics of matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry ( MALD-TOF/TOF ) high-energy collision induced dissociation ( CID ) is to produce abundant immonium ( IM ) ions that can offer a wealth of information for peptide composition. However, MALDI-TOF/TOF is generally used for routine protein identification based on database search or de novo sequencing combined with chemical derivation. Consequently, the characteristics of IM ions may not be fully explored and utilized. Here, a total of 239 MS/MS spectra are used to explore the fragmentation features of IM ions with MALDI TOF/TOF spectrometry and their application for peptide identification. IM ion signals can be observed for 14 kinds of amino acids including histidine etc with a positive rate of more than 50%. We have found that the chemical nature of the amino acids and position effects are the two main factors that affect the intensity of fragment ions. In addition, false positive IM ions are mainly derived from Arg, Lys, Leu and Ile residues or mixture peptides. Besides the compositional information, partial sequence information can also be obtained by a comparison of the relative intensity of IM ions. These findings are helpful when performing manual interpretations and could be useful for improving current peptide search algorithms.

La proteomica n la era postgenómica / The Proteomics In The Postgenomic Era

El principal desafío de la biología moderna es entender la expresión, función y regulación del conjunto completo de proteínas codificadas por un organismo, lo cual describe el objetivo del nuevo campo de la proteómica. Las proteínas son las efectoras del trabajo celular, por ello el estudio de sus perfiles globales de expresión y de sus cambios bajo determinadas condiciones fisiológicas o patológicas, permite entender la red compleja de interacciones en que se basa el funcionamiento de una célula. La electroforesis en dos dimensiones (2D-PAGE) es la técnica central de la proteómica. En la actualidad no existe otro método con la capacidad para resolver simultáneamente miles de proteínas en un solo procedimiento y para detectar modificaciones post y co-traduccionales imposibles de predecir a partir de la secuencia genómica. Sus aplicaciones incluyen el análisis de proteomas, señalización, detección de marcadores de enfermedades y cáncer.

The main challenge of modern biology is to understand the expression, function and regulation of the whole set of proteins codified by an organism, which is the objective of the new field of proteomics. Proteins are the effectors of cellular work and the knowledge of their global expression profiles and changes under physiological and pathological conditions can help us to understand the complex network of interactions involved in cellular function. Two-dimensional electrophoresis (2-DE) is the central technology in proteomics. At present no other technique has the throughput and high resolution of 2-DE for the separation of thousands of proteins in one procedure and for the analysis of post-and co-translation modifications, not predictable from the genome sequence. The scope of applications extends from proteome analysis, to cell signaling, disease markers and cancer.

N-Terminal Cyclization of Peptides in LargE-scale Protein Identification Based on Biological Mass Spectrometry / 分析化学

Biological mass spectrometry has been developed for the largE-scale protein identification. The successful identification of protein in proteomic study is based on an effective match of MS data to the sequence in database. Because of the diversity and heterogeneity of protein modification, the experimental data obtained by mass spectrometry does not match the theoretical value sometimes, which makes about 90 percent or more of the tandem mass spectra not be effectively identified. This has become one of the most important technique issues to be resolved in current proteome research. The N-terminal cyclization of peptides, as one of a variety of modification introduced in sample preparation, has been preliminarily studied in this work. The result showed that N-terminal cyclization occurred at the most of the glutamine(Q) or carbamoylmethyl-cysteine(CAM_C) residues and the reaction is often incomplete or partial, both types of peptides could often exist in its respective state at the same time, and the behavior of modified peptides in revered phase chromatography is also changed. The success rate of protein identification could be obviously improved if adding the N-terminal cyclization modification in the database searching. These results will be very helpful in the mass spectrometric data analysis of proteomic study.

Characterization of Recombinant Human Necrosis Factor-α ReceptorⅡ:IgG Fc Fusion Protein by Nano Ultra-high Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry Tandem Mass Spectrometry / 分析化学

The Nano ultra-high performance liquid chromatography-electrospray ionization mass spectrometry tandem mass spectrometry(UPLC-ESI-MS/MS) was used to characterize the primary protein. Samples were digested by trypsin, followed by liquid chromatography-tandem mass spectrometry analysis and database search. Five peptides matched with tumor necrosis factor receptor and seven peptides matched with human IgG1 fragment of the Fc. Further analysis demonstrated that seven peptides all matched with the IgG1 Fc but only partly peptides matched with the other subtypes. RhTNFR:Fc is fused by IgG1 Fc but not other subtypes.

Expression and purification of human S100A2-GST fusion protein in prokaryotic cells / 重庆医科大学学报

Objective:To obtain human S100A2-GST fusion protein for further research on human S100A2(hS100A2)protein function and its interactions with other proteins and systems.Methods:hS100A2 gene from pHAHA-hS100A2 was subcloned into an expression vector pGST-moluc to construct pGST-moluc-hS100A2.The new plasmid was identified by digestion with XhoⅠand EcoRⅠ.The recombinant BL21 was induced with IPTG to express recombinant fusion protein hS100A2-GST,which was purified by Glutathion-Sepharose 4B ball beads,identified by SDS-PAGE and Western Blot,and quantified by Bradford method.Results:After the digestion,the recombinant plasmid was cutted into two fragments,300 bp and 5kb.After treated with IPTG,the recombinant BL21 strain expressed a protein,which was about 36 kD and was recognized specifically by an-ti-hS100A2.Its yield was 5 mg/L bacterial culture.After isolated by Glutathion-Sepharose 4B ball beads,its purity was 92%.Conclusion:hS100A2-GST expression plasmid pGST-moluc-hS100A2 was constructed successfully.hS100A2-GST fusion pro-tein could be expressed in Escherichia coli with higher yield and isolated with higher purity,which lays the foundation for the follow-up of the hS100A2 research.

Methods and Strategies of Novel Proteins Identification in Proteomics / 生物化学与生物物理进展

The combination of tandem spectrometry and database searching is one of the most popular technologies for protein identification.However,only those proteins in the searching database could be identified,and current database is far from completeness.So it is necessary to mining the MS/MS data comprehensively,in which novel protein identification is the most important one.The definition of novel protein could be divided into three levels according to their annotations of sequences and functions.As a part of protein identification,the main approaches used to identify novel protein are basing on the following two different ways:de novo sequencing combined with similarity search and searching against nucleotide acid databases such as EST or genome databases.Several mature or newly developed methods and techniques were summarized,and the problems and strategies discussed here would be helpful for the related researches.

Expression,Purification and Identification of Recombinant Human Neuroglobin / 中国生物工程杂志

Neuroglobin(NGB),widely and specially expressed in neurons of various vertebrates,was reported to be a scavenger of reactive oxygen species and/or a stress-responsive sensor for neuroprotection of hypoxic and ischemic insults.However,the underlying mechanism remained unknown.It is important for the functional study of NGB by preparing the protein samples.To address it,the human neuroglobin cDNA fragment was amplified by RT-PCR from human fetus brain and cloned into the prokaryotic expression vector pBV220,and then transformed into E.coli HB101 cells for expression.The expressed protein was purified by gel filtration and anion exchange column followed with SDS-PAGE and Western blot identification,and then desalting by sephadex G-25 medium.The prepared neuroglobin was further identified by mass spectrometry and N-terminal amino acid sequencing analysis.The expressed bacterium,the lysate supernatant and the purified protein samples had visible red color,showing a typical activity of the globin family proteins.In conclusion,the neuroglobin was not only expressed in soluble form with high-efficiency in E.coli,but could also be easily purified with only two steps.The preparation of the NGB proteins will advance the neuroprotective function and mechanism studies of the novel globin.