Improved mass spectrometric identification of gel-separated hydrophobic membrane proteins after sodium dodecyl sulfate removal by ion-pair extraction

Separation and identification of hydrophobic membrane proteins is a major challenge in proteomics. Identification of such sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins by peptide mass fingerprinting (PMF) via matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) is frequently hampered by the insufficient amount of peptides being generated and their low signal intensity. Using the seven helical transmembrane-spanning proton pump bacteriorhodopsin as model protein, we demonstrate here that SDS removal from hydrophobic proteins by ion-pair extraction prior to in-gel tryptic proteolysis leads to a tenfold higher sensitivity in mass spectrometric identification via PMF, with respect to initial protein load on SDS-PAGE. Furthermore, parallel sequencing of the generated peptides by electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) was possible without further sample cleanup. We also show identification of other membrane proteins by this protocol, as proof of general applicability.     

A thermostable alpha-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Comparative proteome analysis of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> with pXO1 plasmid content

Bacillus anthracis the causative agent of anthrax, is an important pathogen among the Bacillus cereus group of species because of its physiological characteristics and its importance as a biological warfare agent. Tripartite anthrax toxin proteins and a poly-D-glutamic acid capsule are produced by B. anthracis vegetative cells during mammalian hosts infection and when cultured in conditions that are thought to mimic the host environment. To identify the factors regulating virulence in B. anthracis the whole cell proteins were extracted from two B. anthracis strains and separated by narrow range immobilized pH gradient (IPG) strips (pH 4–7), followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins that were differentially expressed were identified by the peptide fingerprinting using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 23 proteins were identified as being either upregulated or downregulated in the presence or absence of the virulence plasmid pXO1. Two plasmid encoded proteins and 12 cellular proteins were identified and documented as potential virulence factors.     

Proinflammatory exoprotein characterization of toxic shock syndrome Staphylococcus aureus

Pulsed-field gel electrophoresis (PFGE) clonal type USA200 is the most widely disseminated Staphylococcus aureus colonizer of the nose and is a major cause of toxic shock syndrome (TSS). Exoproteins derived from these organisms have been suggested to contribute to their colonization and causation of human diseases but have not been well-characterized. Two representative S. aureus USA200 isolates, MNPE (α-toxin positive) and CDC587 (α-toxin mutant), isolated from pulmonary post-influenza TSS and menstrual vaginal TSS, respectively, were evaluated. Biochemical, immunobiological, and cell-based assays, including mass spectrometry, were used to identify key exoproteins derived from the strains that are responsible for proinflammatory and cytotoxic activity on human vaginal epithelial cells. Exoproteins associated with virulence were produced by both strains, and cytolysins (α-toxin and γ-toxin), superantigens, and proteases were identified as the major exoproteins, which caused epithelial cell inflammation and cytotoxicity. Exoprotein fractions from MNPE were more proinflammatory and cytotoxic than those from CDC587 due to high concentrations of α-toxin. CDC587 produced a small amount of α-toxin, despite the presence of a stop codon (TAG) at codon 113. Additional exotoxin identification studies of USA200 strain [S. aureus MN8 (α-toxin mutant)] confirmed that MN8 also produced low levels of α-toxin despite the same stop codon. The differences observed in virulence factor profiles of two USA200 strains provide insight into environmental factors that select for specific virulence factors. Cytolysins, superantigens, and proteases were identified as potential targets, where toxin neutralization may prevent or diminish epithelial damage associated with S. aureus.     

Identification of p21Cip1 binding proteins by gel electrophoresis and capillary liquid chromatography microelectrospray tandem mass spectrometry

Proteins bound to a glutathione-S-transferase-p21Cip1 affinity column were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified using tandem mass spectrometry. Capillary liquid chromatography coupled to microelectrospray tandem mass spectrometry (capLC-microESI MS/MS) in an ion trap allowed identification of the proteins present in the gel bands. Of eleven bands analyzed, fifty-three proteins were identified. More than one hundred tryptic peptides were detected on-line, automatically fragmented and used for protein characterization in databases. Samples were also analyzed by off-line nanospray and matrix-assisted laser desorption/ionization mass spectrometry. CapLC-microESI MS/MS was the most efficient technique for the analysis of these protein mixtures.     

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.     

Binding of lithium dodecyl sulfate to polyacrylamide gel at 4 degrees C perturbs electrophoresis of proteins

Although polyacrylamide gel has no affinity to lithium dodecyl sulfate (LDS) at 25 degrees C, the gel maximally binds 17 mg of LDS per gram dry weight at 4 degrees C. When polyacrylamide gel electrophoresis is carried out at 4 degrees C in the presence of LDS instead of sodium dodecyl sulfate (SDS) using a continuous buffer system, migration of proteins with lower molecular weight is accelerated as a result of the deficiency of LDS in the frontal region of the gel. When the gel is saturated with LDS, electrophoresis in the presence of LDS at 4 degrees C shows a resolution higher than that of SDS-polyacrylamide gel electrophoresis at 25 degrees C.     

Purification and characterization of an extracellular exochitinase,beta-N-acetylhexosaminidase,from the fungal mycoparasite Stachybotrys elegans

The mycoparasite Stachybotrys elegans produces two exo- and one endo-acting chitinases when grown on chitin. We purified to homogeneity one of the exo-acting chitinases, beta-N-acetylhexosaminidase and partially characterized its physical and biochemical properties. The native enzyme has a molecular mass of 120 kDa when determined by gel filtration and 68 kDa by sodium dodecyl sulfate - polyacrylamide gel electrophoresis indicating that the native protein probably occurs as a dimer in solution. The purified beta-N-acetylhexosaminidase is most active at pH 5.0 and 40 degrees C and hydrolyzes the p-nitrophenyl-N-acetyl-beta-D-glucosaminide with apparent Km of 84.6 microM. Polyclonal antibodies raised against the 68-kDa beta-N-acetylhexosaminidase (NAG-68) indicated that the antibody is highly specific and recognizes the protein in crude filtrate preparation. This suggests that the protein is a not a proteolytic product of another protein. Western blot analysis showed that the activity of NAG-68 was induced when S. elegans was grown on purified cell wall fragments of its host, Rhizoctonia solani, as well as during antagonistic interaction of the mycoparasite and host when both were grown on synthetic medium with or without supplemental carbon source.     

The role of glucose in the Kluyveromyces bulgaricus flocculation phenomenon: transduction by cAMP-dependent protein kinase pathway?

The lipopolysaccharide (LPS) from eight strains of Yersinia pestis which had been cultured at 28 degrees C appeared to be devoid of an O-antigen when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LPS isolated from three of these strains which had been cultured at 37 degrees C also appeared to be devoid of an O-antigen. When the LPS from Y. pestis strain CO92 was purified and analysed by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry, the observed signals were in the mass range predicted for molecules containing lipid A plus the core oligosaccharide but lacking an O-antigen. The nucleotide sequence of Y. pestis strain CO92 revealed the presence of a putative O-antigen gene cluster. However, frame-shift mutations in the ddhB, gmd, fcl and ushA genes are likely to prevent expression of the O-antigen thus explaining the loss of phenotype.     

Isolation of a novel protein involved in the transport of fructose by an inducible phosphoenolpyruvate fructose phosphotransferase system in Streptococcus mutans.

Fructose transport in Streptococcus mutans LG-1 is mediated by at least two distinct phosphoenolpyruvate fructose phosphotransferase systems. One system is constitutive and consists of membrane components enzyme II as well as enzyme I and heat-stable protein. The other system is inducible and requires, in addition to enzyme I and heat-stable protein, a soluble substrate-specific protein for catalytic activity. This protein factor, designated IIIfru, was purified by DEAE-cellulose chromatography, hydroxylapatite chromatography, molecular sieving on Sephadex G-75, and preparative electrophoresis. The purified preparation showed only one protein band, with a molecular weight of 12,600, on sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, on gel electrophoresis with the discontinuous buffer Tris-glycine, and after electrofocusing in gel (pI congruent to 3.7). The molecular weight of the native protein determined by gel filtration at 4 degrees C was 51,000. Immunodiffusion experiments performed with immunoglobulins prepared against the purified IIIfru from S. mutans LG-1 suggested that other S. mutans strains possessed a IIIfru. No precipitin bands, however, were detected with extracts from S. salivarius, S. sanguis, S. lactis, S. faecalis, Staphylococcus aureus, Bacillus subtilis, Lactobacillus casei, and Escherichia coli.     

Targeting of porin to the outer membrane of Escherichia coli. Rate of trimer assembly and identification of a dimer intermediate

Porin, a transmembrane protein in the outer membrane of Escherichia coli, exists in a trimeric structure which is not dissociated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 25 degrees C. This unusual stability was utilized in the study of the conformational changes which accompany the targeting of porin to the outer membrane. A delay of 16-44 s between completion of synthesis of a monomer and its assembly into a trimer was found from the ratio of monomers to trimers found in exponentially growing cells. Pulse-chase experiments showed that rapid processing of precursor OmpF molecules was followed by assembly into sodium dodecyl sulfate-resistant oligomers with a half-time of 20 s at 30 degrees C. An intermediate in assembly was isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis below 10 degrees C and was identified as a metastable dimer.     

Matrix-assisted laser desorption/ionization time of flight mass spectrometry peptide mass fingerprints and post source decay: a tool for the identification and analysis of phloem proteins from Cucurbita maxima Duch. separated by two-dimensional polyacrylamide gel electrophoresis

A combination of gel electrophoresis and mass spectrometry was used to analyze the soluble proteins from phloem sap of Cucurbita maxima Duch. Phloem proteins were separated using two-dimensional gel electrophoresis. Coomassie-stained spots were cut out and subjected to tryptic digestion. To identify proteins, peptide mass fingerprints were determined by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. In addition, MALDI-TOF post source decay measurements were used to obtain partial sequence information for the proteins. Results from both approaches were used for database searches. In this study, 17 proteins in the mass range 5-50 kDa were analyzed. Of these proteins six could be clearly identified, seven showed significant homologies to known plant proteins, and four were not significantly homologous to database entries. The present study suggests that the applied method is feasible for a large-scale analysis and identification of phloem proteins derived from different organs or from plants kept under various physiological conditions.     

金黄色葡萄球菌拮抗菌株的筛选鉴定及其抗菌物质分析

【背景】植物内生菌的次生代谢产物是新型天然活性物质的重要来源。【目的】从芍药内生细菌中筛选对金黄色葡萄球菌有抑菌活性的菌株和次生代谢产物。【方法】采用平板对峙法筛选拮抗菌株,根据形态学特征和分子生物学的方法鉴定菌株,PCR扩增检测合成脂肽类物质的功能基因;运用牛津杯法依次测定内生细菌发酵液和脂肽类粗提物的抑菌活性,利用Sephadex LH-20凝胶层析分离脂肽类物质,利用基质辅助激光解吸电离飞行时间质谱分析具有抑菌作用的分离组分。【结果】共筛选出13株对金黄色葡萄球菌具有不同程度抑制作用的内生菌株,其中菌株SY11的抑菌作用最为显著,其发酵液和脂肽类粗提物均具有较强的抑制作用。结合形态学鉴定以及16S r RNA基因序列分析,鉴定其为解淀粉芽孢杆菌(Bacillusamyloliquefaciens)。PCR扩增检测表明菌株SY11含有3个合成脂肽类物质的功能基因fenA、ituD和srfkn,推测该菌株可能具有合成脂肽类物质的能力。根据具有抑菌活性分离组分的质谱分析结果,推测其有效物质的主要成分为Bacillomycin D。【结论】解淀粉芽孢杆菌SY11对金黄色葡萄球菌有良好抑制效果,其脂肽类粗提物也具有较强的体外抑菌活性。本研究为芍药内生细菌的开发应用奠定了基础。     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

A proteomic approach for investigation of photosynthetic apparatus in plants

The proteome of the photosynthetic apparatus of barley (Hordeum vulgare), obtained by analysis of thylakoids without any previous fractionation, was mapped by native electrophoresis followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as the second dimension two-dimensional-blue native (2-D/BN)/SDS-PAGE). This protocol provided an excellent alternative to the 2-D-isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 2-D separation of the most hydrophobic thylakoid proteins. Monocots and dicots showed significant differences in the first dimension while in the second dimension patterns appeared similar. Identification of each spot was performed by internal peptide primary sequence determination using both nano-electrospray ionization tandem mass spectrometry and, to a lesser extent, peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight using MALDI-TOF. This is due in particular to the fact that a limited number of peptides was obtained after trypsin digestion of these highly hydrophobic proteins. A larger number of peptides from hydrophilic intermembrane domains of transmembrane proteins were detected. Despite this, about 70% of the expected proteins were identified, including proteins with grand average of hydropathicity scores higher than 0.5. It is therefore reasonable to assert that protein hydrophobicity is not the limiting factor. Small proteins were not well identified with trypsin digestion. Instead some of these could be identified using acid hydrolysis. The method presented here does not require prefractionation of different thylakoid complexes and consequently gives confidence in comparing the proteome of the photosynthetic apparatus before and after treatment. It thus allows us to understand the molecular mechanisms underlying physiological adaptations of higher plants and to perform screening of photosynthetic mutants.     

Reovirus major capsid protein expressed in Escherichia coli

A DNA copy of the open reading frame of the S4 gene of reovirus type 3 was cloned into a temperature-regulated bacterial expression vector. Induction at 42 degrees C resulted in the synthesis of a polypeptide that comigrated with virion capsid protein sigma 3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with sigma 3-specific antisera. The protein was expressed in bacteria as insoluble aggregates that accumulated in polar inclusion bodies. Aggregated product also resulted when the expression system was manipulated to induce bacterial sigma 3 (b sigma 3) synthesis at temperatures below 42 degrees C. Various methods used to solubilize b sigma 3 did not yield the monomeric protein. The results indicate that sigma 3, the major surface component of reovirions, is expressed in transfected Escherichia coli as an aggregated, disulfide cross-linked protein.     

Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究

由本实验室筛选得到的摩尔摩根氏菌J-8菌株可将底物1-苯基-2-甲氨基丙酮专一性地转化为d-伪麻黄碱。以M.morganiiJ-8为出发菌株,菌体超声破碎后,经硫酸铵沉淀、Phenyl Superose疏水柱层析、DEAD阴离子柱层析和非变性凝胶电泳四步纯化获得电泳纯羰基不对称还原酶。亚基分子质量为42.5kD,高效液相色谱分析酶的分子质量约为84.1kD,初步认为该酶为二聚体蛋白。对所得到的部分纯化酶的酶学性质做了初步研究,纯酶进行基质辅助激光解析电离-飞行质谱分析,比对结果显示为与亮氨酸脱氢酶蛋白有很高相似性。