基质辅助激光解析电离-串联飞行时间质谱与电喷雾离子化-四极杆-飞行时间质谱分析蛋白质

为比较基质辅助激光解析电离-串联飞行时间质谱(MALDI-TOF-TOF)和电喷雾离子化-四极杆-飞行时间质谱(ESI-Q-TOF)在蛋白质定性和定量分析方面的性能,分别利用两种仪器对用于相对和绝对定量的等量异位标签(iTRAQ)标记的雌激素刺激前后MCF7细胞内的蛋白质水解产生的多肽进行了定性和定量分析.结果表明,用MALDI-TOF-TOF和ESI-Q-TOF方法分别鉴定出1086种和848种蛋白质,其中相同的蛋白质为633种;MALDI-TOF-TOF和ESI-Q-TOF方法分别找到38种和33种表达差异在0.5倍以上的蛋白质,其中3种为相同的蛋白质.实验数据说明,两种仪器在蛋白质定性和定量分析方面有很大的互补性,将两种仪器结合使用,不仅能够显著提高蛋白质定性和定量分析的覆盖率,而且可提高分析的置信度.     

Metal labeling for accurate multiplexed peptide quantification via matrix-assisted laser desorption/ionization mass spectrometry

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.     

Production and Characterization of a Milk-clotting Protease Produced in Submerged Fermentation by the Thermophilic Fungus Thermomucor indicae-seudaticae N31

Proteases are some of the most important industrial enzymes, and one of their main applications is for the production of cheese in the dairy industry. Due to a shortage of animal rennet, microbial coagulant proteases are being sought. In this work, the production of microbial rennet from Thermomucor indicae-seudaticae N31 was studied in submerged fermentation. The best enzyme production was obtained in a fermentation medium containing 4 % wheat bran as the substrate in 0.3 % saline solution, incubated for 72 h at 45 °C and 150 rpm. The value of the milk clotting activity (MCA) was 60.5 U/mL, and the ratio to proteolytic activity (MCA/PA) was 510. The crude enzyme showed optimum pH at 5.5 and two peaks of optimum temperature (MCA at 65 °C and PA at 60 °C). The MCA was stable in the pH range 4.0–4.5 for 24 h and up to 55 °C for 1 h. It was stable during storage at different temperatures (?20 to 25 °C) for 10 weeks. Based on these results, we conclude that microbial rennet from T. indicae-seudaticae N31 produced by submerged fermentation showed good prospects of replacing traditional rennet.     

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected.     

Radiation effects of paraffins as polymer model compounds—II. Crosslinking determination by liquid chromatography

The G-values of crosslinking and chain scission of normal paraffins (C₂₀H₄₂, C₂₁H₄₄, C₂₃H₄₈ and C₂₄H₅₀) and squalane (C₃₀H₆₂) as the model compounds of polymers were determined by the measurements with a liquid chromatography (LC) after Co-60 γ-rays irradiation in solid and in liquid phases at -77, 25 and 55°C. The crosslinking products as dimer, trimer and the more polymeric products were well resolved and the relative yield of polymeric products increased with increasing dose. The G(cross-linking) of n-paraffins was 1.0 in the crystalline at -77°C and 1.15 in the crystalline at 25°C, and 1.7–1.8 in the liquid phase at 55°C of irradiation temperature. The G(crosslinking) of squalane was 1.3 in the glassy phase at -77°C and 1.6 in the liquid phase at 25 and 55°C. However, the chain scission products were too small to detect by the LC measurement, that is, the yield was estimated to be less than 1/10 of the crosslinking products.The unsaturations which appeared in u.v. detector of LC spectrum were observed in monomer, dimer, trimer and the more polymeric products, and the relative concentration was the higher in the more polymeric products, especially in liquid phase. So the molecules with unsaturation would react preferentially with other molecules as monomer or dimer to produce the polymeric products including the unsaturation.     

Aliphatic dipeptide tags for multi-2-plex protein quantification

Mass-balanced (1)H/(2)H-isotope dipeptide tag (MBIT) is diversified as aliphatic tags for multiplexed protein quantification. Aliphatic MBITs are based on the N-acetyl-Xxx-Ala dipeptide, where Xxx is an artificial amino acid with a linear alkyl side chain from C(2)H(5) to C(8)H(17) (C(2)-C(8) tags). (1)H/(2)H isotopes are encoded in the methyl groups of N-acetyl and Ala to yield a pair of isobaric tags with 2-plex quantitation signals separated by 3 Da. C(2)-C(5) tags are prepared by solid-phase synthesis, while C(6)-C(8) tags are synthesized by olefin metathesis in solution. These aliphatic tags are made reactive toward the primary amines of peptides, and the relative abundances of quantitation signals are characterized using both matrix-assisted laser desorption ionization and electrospray ionization tandem mass spectrometry. MBIT-linked peptides co-migrate in reverse-phase liquid chromatography (LC), and their tandem mass spectra exhibit 2-plex quantitation signals as well as sequence ions in similar abundances. As the length of alkyl side chain increases, C(2)-C(8) tags show a stepwise increase in both the LC retention time and the relative abundance of quantitation signals. In addition, the quantitation linearity is well-maintained in a 15-250 fmol range. The multiplexing capability of aliphatic MBITs is demonstrated by applying three different tags (C(6)-C(8) tags) to the quantification of yeast heat shock proteins expressed under four different physiological conditions.     

Purification and Characterization of Tyrosine Phenol Lyase from Citrobacter freundii

The purification and characterization of intracellular tyrosine phenol lyase from Citrobacter freundii has been carried out. The enzyme was purified 35-fold to homogeneity by ammonium sulphate precipitation and hydrophobic interaction chromatography. Its subunit molecular weight was found to be 52 kDa on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified tyrosine phenol lyase showed maximum activity in borate buffer (0.05 M at pH 8.5) at 45 °C after 20 min of incubation. The K _m and V _max values of purified enzyme were found to be 0.446 mm and 0.342 mM/min/mg. This enzyme exhibits t _1/2 of 10, 52 and 130 min at 55, 45 and 35 °C, respectively. The N-terminal amino acid sequence was determined as MET-ASN-TYR-PRO-ALA-GLU-PRO-PHE-ARG-ILE-TRP-TRP-VAL-GLY.     

同系物标记结合凝胶电泳和质谱技术用于蛋白质N端肽鉴定及相对定量

通过酸酐同系物标记蛋白质的N-末端,经过凝胶电泳分离,使用基质辅助激光解析电离飞行时间质谱仪进行鉴定,建立了一种基于化学标记和质谱技术的、用于蛋白质的N-末端鉴定和相对定量新方法。标记后的蛋白质的N端肽以相差14 Da的成对峰形式在质谱中出现,而非N-末端肽则是以单峰呈现,从而实现了末端肽的特异性识别;通过端肽的信号强度对3种标记蛋白质的不同比例的混合溶液实现了相对定量分析。     

Thermal Stability of α-Phycoerythrocyanin

The α-subunit of the phycobiliprotein, phycoerythrocyanin (α-PEC), from Mastigocladus laminosus shows photoreversible photochromism that is based on the Z ↔ E isomerization of the phycoviolobilin (PVB) chromophore. Thermal stability of the photochemistry and chromoprotein secondary structure have been studied by absorption and circular dichroism (CD) spectroscopy. Both photoisomers are stable and photoconvertible up to ∼ 70 °C. At T > 75 °C, photochemistry ceases because the E-state reverts rapidly thermally to the Z–state. The chromoprotein melts at 72 °C, the apoprotein already at 55 °C, indicating a considerable stabilization of the protein secondary structure by the chromophore.     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

Targeted data acquisition for improved reproducibility and robustness of proteomic mass spectrometry assays

Quantitative mass spectrometry-based proteomic assays often suffer from a lack of robustness and reproducibility. We here describe a targeted mass spectrometric data acquisition strategy for affinity enriched subproteomes—in our case the kinome—that enables a substantially improved reproducibility of detection, and improved quantification via isobaric tags. Inclusion mass lists containing m/z, charge state, and retention time were created based on a set of 80 shotgun-type experiments performed under identical experimental conditions. For each target protein, peptides were selected according to their frequency of observation and isobaric tag for relative and absolute quantitation (iTRAQ) reporter ion quality. Retention times of selected peptides were aligned using similarity driven pairwise alignment strategy yielding <1 min standard deviation for 4 h gradients. Multiple fragmentation of the same peptides resulted in better statistics and more precise reporter ion based quantification without any loss in coverage. Overall, 24% more target proteins were quantified using the targeted data acquisition approach, and precision of quantification improved by >1.5-fold. We also show that a combination of higher energy collisional dissociation (HCD) with collisional induced dissociation (CID) outperformed pulsed-Q-dissociation (PQD) on the OrbitrapXL. With the CID/ HCD based targeted data acquisition approach 10% more quantifiable target proteins were identified and a 2-fold increase in quantification precision was achieved. We have observed excellent reproducibility between different instruments, underlining the robustness of the approach.     

Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00?±?0.15 and 0.21?±?0.02 U/ml were obtained for xylanase and β-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly β-xylosidase. The optimal temperature and pH of the xylanase activity were 75°C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for 7 days at pH?7.0–8.0. The half-lives of the enzyme were 4 h at 80°C, 2 h at 90°C, and 1 h at 100°C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and β-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.     

Electron-Capture and -Transfer Dissociation of Peptides Tagged with Tunable Fixed-Charge Groups: Structures and Dissociation Energetics

Pyridiniummethylcarbonyl moieties that were previously designed on the basis of electronic structure analysis are now utilized as fixed-charge tags with tunable electronic properties to be used for N-terminal peptide derivatization and sequencing by electron-transfer dissociation. Dipeptides AK and KA were derivatized at the peptide N-terminus with 4-dimethylaminopyridinium-N-acetyl (DMAP-ac) and pyridinium-N-acetyl (pyrid-ac) tags of increasing intrinsic recombination energies. Upon the capture of a free electron or electron transfer from fluoranthene anions, (DMAP-ac-AK+H)²⁺, (DMAP-ac-KA+H)²⁺, (pyrid-ac-AK+H)²⁺ and (pyrid-ac-KA+H)²⁺ ions, as well as underivatized (AK+2H)²⁺, completely dissociated. The fixed-charge tags steered the dissociation upon electron transfer to form abundant backbone N–C_α bond cleavages, whereas the underivatized peptide mainly underwent H-atom and side-chain losses. Precursor ion structures for the tagged peptides were analyzed by an exhaustive conformational search combined with B3LYP/6-31+G(d,p) geometry optimization and single-point energy calculations in order to select the global energy minima. Structures, relative energies, transition states, ion–molecule complexes, and dissociation products were identified for several charge-reduced species from the tagged peptides. The electronic properties of the charge tags and their interactions with the peptide moieties are discussed. Electrospray ionization and electron-transfer dissociation of larger peptides are illustrated with a DMAP-tagged pentapeptide.     

Dyed polyvinyl chloride films for use as high-dose routine dosimeters in radiation processing

Characteristics of the polyvinyl chloride (PVC) films containing 0.11 wt% of malachite green oxalate or 6GX-setoglausine and about 100 μm in thickness were studied for use as routine dosimeters in radiation processing. These films show basically color bleaching under irradiation with ⁶⁰Co γ-rays in a dose range of 5–50 kGy. The sensitivity of the dosimeters and the linearity of dose-response curves are improved by adding 2.5% of chloral hydrate [CCl₃CH(OH)₂] and 0.15% hydroquinone [HOC₆H₄OH]. These additions extend the minimum dose limit to 1 kGy covering dosimetry requirements of the quality assurance in radiation processing of food and healthcare products. The dose responses of both dyed PVC films at irradiation temperatures from 20°C to 35°C are constant relative to those at 25°C, and the temperature coefficients for irradiation temperatures from 35°C to 55°C were estimated to be (0.43±0.01)%/°C. The dosimeter characteristics are stable within 1% at 25°C before and 60 days after the end of irradiation.     

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so‐called cysteine‐reactive covalent capture tags (C³T), for the isolation of Cys‐containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine‐containing peptides. The combination of C³T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

The application of 2,4,6-trinitrobenzene-1-sulphonic acid in the differential pulse polarographic determination of amines

The differential pulse polarographic behaviour of 2,4,6-trinitrophenyl (TNP) derivatives of several primary amines and amino acids was investigated in the presence of sulphite ion. All the derivatives produced a polarographic peak for their complexes with sulphite (1 × 10^?2 M) in pH 8.0 phosphate buffer (0.05 M)/0.1 M potassium chloride. The derivatives of proteins and peptides did not give such a peak. A 5-min reaction time at room temperature (or 50°C for lysine) and pH 10.5 using 1 × 10^?4 M 2,4,6-trinitrobenzene-1-sulphonic acid provides the optimal conditions for the determination of 5 × 10^?6?2.5 × 10^?5 M amines. The relative standard deviation for determining 1 × 10^?5 M glycine (n = 5) was 1%.     

Studying the effect of microwave heating on the digestion process and identification of proteins

The impact of microwave irradiation on the in‐solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI‐MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC‐MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave‐assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.