A rapid method for isolation and purification of an anticoagulant from Whitmania pigra

Whitmania pigra is common in China and has been used as a traditional Chinese anticoagulant medicine for years, but its effective components are unknown to scientists. In this article we report a rapid method for isolation and purification of an anticoagulant from W. pigra for the first time. An acetone-water extract of W. pigra was subjected to anion-exchange chromatography on a Sephadex DEAE A-50 column, and gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns successively, which afforded a fraction with potent anticoagulant activity. An anticoagulant was isolated and purified from this fraction by reversed-phase high-performance liquid chromatography (RP-HPLC). It was identified as a single pure substance by RP-HPLC and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This component was named whitmanin and its molecular weight was determined as 8608 Da by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS).     

亲和层析结合生物质谱技术分析鉴定猕猴血清中的重组人内皮抑制素

建立了将固相亲和层析与生物质谱技术相结合 ,分离提取鉴定给药猕猴血清中含有 6×His序列的重组人内皮抑制素的方法。用固相免疫亲和层析和金属螯合层析两种方法分别提取并富集血清中的重组药物 ,采用基质辅助激光解吸 电离飞行时间质谱的直接分析和肽质量指纹谱分析与数据库检索 ,分别对两种亲和提取方法得到的药物蛋白前体collagenXVIII[Homosapiens](IDofSWISS -PROT TrEMBL :Q8WXI5 )进行了鉴定     

Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.     

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.     

Optimization of microfabricated nanoliter-scale solid-phase extraction device for detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization mass spectrometry

A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins.     

Trends in metal-binding and metalloprotein analysis

This review describes recent tendencies for metal-binding and metalloprotein analysis, emphasizing metal quantification in proteins through X-ray, atomic absorption, mass spectrometric techniques, and others. Hyphenated techniques such as capillary electrophoresis-synchrotron radiation X-ray fluorescence (CE-SRXRF), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF-MS), etc. are also presented. As protein separation techniques electrophoresis (mainly sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE), capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are indicated, due to their inherent sensitivity, resolution and/or easy implementation. Latest challenges in metallomics are also commented.     

小分子表面活性剂在胶内酶切增效中的应用

本研究成功地将一种有机小分子表面活性剂RapiGest SF(Waters)用于改进电泳分离的蛋白质的鉴定效率。通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)的肽质量指纹谱,考察了酶切时间、加入量、加样次序、点靶方法对方法灵敏度、蛋白质鉴定率的影响。RapiGest SF浓度为0.5%~1%,在酶切之前加入可获得更多的肽段峰和更高的鉴定率。本方法考染体系的总灵敏度为332fmol,银染体系为664fmol。比较了RapiGest SF与MALDI-TOF-MS和电喷雾质谱(ESI-MS)兼容性,未观察到明显的负影响。方法操作简便,重复性较好,适合鉴定电泳分离的低丰度蛋白质。     

Properties of mouse vomeronasal receptor and assessment of its role in pheromone signalling

Vomeronasal type 2 receptor (V2Rx) from Swiss mouse (Mus musculus (L.)) was analyzed by high-resolution ion-exchange chromatography, reversed-phase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), Ion Spray tandem mass spectrometry (MS/MS), 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 1-aminoanthracene (1-AMA) fluorometric assay. Vomeronasal sensory neuronal cell bound proteins were resolved into major protein peaks. Several proteins were identified and subsequently purified as the V2Rx receptor on 10% SDS-PAGE with trace amounts of other protein bands. The molecular weight of the identified V2Rx was 109 kDa. MALDI-TOF and micro-sequencing experiments demonstrated that the identified V2Rx receptor shared considerable sequence similarity with vomeronasal receptor type 2 (NCBI Accession Number AB267725), which is a seven transmembrane peptide with 912 amino acid residues. The molecular characterization revealed that the N-terminus of the V2Rx receptor contained the 11GAEAAE16 domain involved in pheromone signalling. The biometric assay (octanamine-V2Rx binding) showed the identified V2Rx receptor and mouse sex pheromone to 2-octanamine (methyl heptyl) in a 1:1 ratio. Uptake of odourants determined in physiological condition showed enhanced V2Rx receptors as volatile hydrophobic pheromone receptors in the vomeronasal neuron of the Swiss mouse.     

Comparison of planar SDS-PAGE,CGE-on-a-chip,and MALDI-TOF mass spectrometry for analysis of the enzymatic de-<Emphasis Type="Italic">N</Emphasis>-glycosylation of antithrombin III and coagulation factor IX with PNGase F

Three different analytical techniques (planar SDS-PAGE, CGE-on-a-chip and MALDI-TOF-MS) applied for determination of the molecular weight of intact and partly and completely de-N-glycosylated human serum glycoproteins (antithrombin III and coagulation factor IX) have been compared. N-Glycans were removed from the protein backbone of both complex glycoproteins using PNGase F, which cleaves all types of asparagine-attached N-glycan provided the oligosaccharide has at least the length of a chitobiose core unit. Two of the applied techniques were based on gel electrophoretic separation in the liquid phase while the third technique was the gas-phase technique mass spectrometry. It was demonstrated that the enzymatic de-N-glycosylation generally worked well (completely or partially) with both glycoproteins (one containing only N-glycans and the second N- and O-glycans). All three methods were suitable for monitoring the de-N-glycosylation progress. While the molecular weights determined with MALDI-TOF-MS were most accurate, both gel electrophoretic methods provided molecular weights that were too high because of the attached glycan structures. Figure CGE-on-a-chip, SDS-PAGE and MALDI mass spectrometric pattern obtained from therapeutic glycoprotein     

Purification and characterization of Bacillus cereus protease suitable for detergent industry

An extracellular alkaline protease from an alkalophilic bacterium, Bacillus cereus, was produced in a large amount by the method of extractive fermentation. The protease is thermostable, pH tolerant, and compatible with commercial laundry detergerts. The protease purified and characterized in this study was found to be saperior to endogenous protease already present in commercial laundry detergents. The enzyme was purified to homogeneity by ammonium sulfate precipitation, concentration by ultrafiltration, anionexchange chromatography, and gel filtration. The purified enzyme had a specific activity of 3256.05 U/mg and was found to be amonomeric protein with a molecular mass of 28 and 31 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE, respectively. Its maximum protease activity against casein was found to be at pH 10.5 and 50°C. Proteolytic activity of the enzyme was detected by casein and gelatin zymography, which gave a very clear protease activity zone on gel that corresponded to the band obtained on SDS-PAGE and nondenaturing PAGE with a molecular mass of nearly 31 kDa. The purified enzyme was analyzed through matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and identified as a subtilisin class of protease. Specific serine protease inhibitors, suggesting the presence of serine residues at the active site, inhibited the enzme significantly.     

Characterization of a new methylated beta-cyclodextrin with a low degree of substitution by matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography using evaporative light scattering detection

A new methylated beta-cyclodextrin with a low degree of substitution was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) with evaporative light scattering detection. Using alpha-cyano-4-hydroxycinnamic acid as the matrix and the thin layer method as the deposition procedure, MALDI-TOF-MS revealed that the mixture was composed of CDs bearing from 2 to 8 methyl groups with an average degree of substitution (DS) of 0.7 (i.e. 0.7 methyl groups per glucopyranose unit). Using a Purospher Star RP-18 endcapped column with acetonitrile-water mobile phase in gradient elution mode, HPLC was employed at analytical scale to obtain a chromatographic fingerprint of the crude mixture and at semi-preparative scale to fractionate it. MALDI-TOF-MS of these fractions revealed that the overall retention of the different derivatives, which depicts their polarity, was mainly driven by the DS and increased with the number of methyl groups on the CD moiety.     

富硒螺旋藻光谱特性及其含硒蛋白质组分析

以富硒螺旋藻( Selenium enriched spirulina platensis, Se-SP)为材料,采用红外、紫外-可见吸收光谱分析Se-SP的光谱特征,并利用二维电泳技术(Two-dimensional electrophoresis,2DE)分离、飞行时间质谱(Matrix assisted laser desorption ionization time of flight, MALDI-TOF)鉴定和电感耦合等离子体质谱( Inductively coupled plasma mass spectrometry, ICP-MS)检测硒含量,对Se-SP蛋白质组及其含硒蛋白质分布进行初步鉴定和分析。结果显示,硒对螺旋藻蛋白质组中多种蛋白质表达有一定影响,观察到对捕光色素蛋白如藻蓝蛋白( Phycocya-nin, PC)、别藻蓝蛋白( Allophycocyanin , APC)和 Robisco大亚基及超氧化物歧化酶( Superoxide Dismutase, SOD)等蛋白质具有明显上调作用。硒在Se-SP蛋白质组中存在较为普遍,但分布相对集中在pH 4~7、分子质量14.4~43 kDa的蛋白质中。硒在藻体特定蛋白质中的存在形式还有待深入研究和鉴定。     

Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from <Emphasis Type="Italic">Hypocrea orientalis</Emphasis>

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7–22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19–29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K _m and V _max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.     

High-efficiency protein extraction from polyacrylamide gels for molecular mass measurement by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry

A simple and fast method of protein extraction from Coomassie Brilliant Blue (CBB)-stained polyacrylamide gels suited for molecular mass measurement of proteins by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) is reported. Proteins in CBB-stained gel pieces were extracted by a 10-min soaking in 0.1 M NaOH at 25 degrees C. The recovery of this one-step extraction method was 34-73% for proteins <67 kDa. CBB adduction to proteins during mass spectrometric analysis was avoided by a destaining step before the alkaline extraction. The molecular mass values of the extracted proteins coincided with those of purified proteins within +/-0.01-0.10% deviation for all the proteins <36 kDa. Because of the high extraction recovery, mass measurement was possible for the proteins extracted from CBB-stained gels with loaded protein quantities as little as 34 ng for cytochrome c, alpha-lactalbumin, myoglobin, beta-lactoglobulin, trypsinogen, and carbonic anhydrase (12.4-29.0 kDa), 340 ng for glyceraldehyde-3-phosphate dehydrogenase (35.6 kDa) and albumin (66.3 kDa). This method provides a highly efficient approach to utilize CBB-stained one- or two-dimensional gels for whole protein analysis using MALDI-TOF-MS.     

Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates: a novel two-dimensional liquid chromatography/mass spectrometry method

A novel two-dimensional two-column liquid chromatography/mass spectrometry (LC/MS) technique is described in this work, where chromatofocusing (CF) has been coupled to nonporous reversed-phase (NPS-RP) HPLC to separate proteins from human breast epithelial whole cell lysates. The liquid fractions from NPS-RP-HPLC are readily amenable to direct on-line analysis using electrospray ionization orthogonal acceleration time-of-flight mass spectrometry (ESI-TOFMS). A key advantage of this technique is that proteins can be 'peeled off' in the liquid phase from the CF column according to their isoelectric points (pI) in the first chromatographic separation dimension. The NPS-RP-HPLC column further separates these pI-focused fractions based upon protein hydrophobicity as the second chromatographic dimension. The third dimension involves on-line molecular weight determination using ESI-TOFMS. As a result, this method has the potential to be fully automated. In addition, a 2-D protein map of pI versus molecular weight is generated, which is analogous to a 2-D gel image. Thus, this technique may provide a means to study differential expression of proteins from whole cell lysates.     

Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.     

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains β-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS). BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds. For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H]⁺, of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.     

超高效液相色谱-串联质谱法测定食品中的三氯蔗糖

建立了食品中三氯蔗糖的超高效液相色谱-电喷雾串联质谱(UPLC- MS/MS)测定方法.样品中的三氯蔗糖用水提取,简单除杂后根据样品基质选用HLB柱净化或用沉淀剂去除样品中的脂肪和蛋白质,用甲醇-水定容.经反相色谱柱分离后,采用多反应监测(MRM)负离子模式检测,定性离子对为m/z 395.07/358.90和397....     

高活性燕麦蛋白源ACE抑制肽的制备、纯化及结构鉴定

利用胰蛋白酶水解燕麦蛋白制备了高血管紧张素转化酶(Angiotensin I-Converting Enzyme, ACE)抑制活性的燕麦蛋白酶解物, 分别采用离子交换色谱、凝胶过滤色谱和反相高效液相色谱等分离手段从酶解物中分离出一种新的强活性ACE抑制肽, 其IC50值为77.3 μmol/L; 通过基质辅助激光解析电离飞行时间串联质谱对其进行结构鉴定, 其氨基酸序列为Glu-Gly-Gly-Tyr-Arg.