一种改进的同步辐射X射线荧光原位分析人肝细胞胞质溶胶内金属蛋白分布的方法

对同步辐射X射线荧光分析技术(SBXRF)原位测定蛋白质经十二烷基硫酸铀．聚丙烯酰胺凝胶电泳(SDS-PAGE)分离后条带中微量元素的方法做了重要改进。通过增加一个于胶步骤，使得本底材料对光源的康普顿散射明显减弱，本底信号降低到约为湿胶的10％，从而可以用来测定蛋白条带内比锌轻的微量元素的相对含量。所提出的方法有望成为一种具有广泛应用前景的生物样品中微量元素的种态分析方法。对人肝细胞胞质溶胶内金属蛋白分布的研究结果表明：在15-95kDa的范围内有6个含锌蛋白，相对分子量分别为17．5、20．5、27、35、55及63kDa，其中63kDa蛋白为主要的含锌蛋白；此外至少还有4种分子量分别为20、23、43和83．5kDa的含铁蛋白分布于这个分子量范围内。     

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.     

Selective enrichment of endogenous peptides by chemically modified porous nanoparticles for peptidome analysis

We report the development of a combined strategy for high capacity, comprehensive enrichment of endogenous peptide from complex biological samples at natural pH condition. MCM-41 nanoparticles with highly ordered nanoscale pores (i.e. 4.8nm) and high-surface area (i.e. 751m(2)/g) were synthesized and modified with strong cation-exchange (SCX-MCM-41) and strong anion-exchange (SAX-MCM-41) groups. The modified nanoparticles demonstrated good size-exclusion effect for the adsorption of standard protein lysozyme with molecular weight (MW) of ca. 15kDa; and the peptides with MW lower than this value can be well adsorbed. Step elution of the enriched peptides with five salt concentrations presented that both modified nanoparticles have high capacity and complementarity for peptides enrichment, and the SAX-MCM-41 nanoparticles has obviously high selectivity for acidic peptides with pI (isoelectric point) lower than 4. Large-scale enrichment of endogenous peptides in 2mg mouse liver extract was achieved by further combination of SCX-MCM-41 and SAX-MCM-41 with unmodified MCM-41 nanoparticles. On-line 2D nano-LC/MS/MS was applied to analyze the enriched samples, and 2721 unique peptides were identified in total. Two-dimensional analysis of MW versus pI distribution combined with abundance of the identified peptides demonstrated that the three types of nanoparticles have comprehensive complementarity for peptidome enrichment.     

Fish muscle parvalbumins as marker proteins for native and urea isoelectric focusing

An isoelectric point (pI) calibration kit containing fish muscle parvalbumins was prepared and tested for its suitability for isoelectric focusing (IEF) in the presence of 8 M urea. The pattern obtained by urea CleanGel IEF consisted of nine bands covering the pI range 4.96-5.64. This range is relevant for species identification of heated fish by urea IEF. The kit may also be used for native IEF in the low pH range, as demonstrated by running an extract made from the kit together with water-soluble fish muscle proteins on Servalyt Precotes 3-6.     

Mass spectrometric characterization of low-molecular-mass color pI markers and their use for direct determination of pI value of proteins

The use of low-molecular-mass color pI markers for the determination of pI values of proteins in gel isoelectric focusing (IEF) in combination with mass spectrometry is described. Different types of substituted phenols of known pI values within the mass range 250-400 were used here as pI markers. The pure, synthesized pI markers were studied by MALDI-TOF/TOF MS. Fragmentation studies of the pI markers were also performed. Only stable and well-characterized pI markers were used in this work. The selected pI markers were mixed with proteins, deposited on a gel and separated in a pH gradient. Color pI markers enable supervision of progress of the focusing process and also estimation of the position of the invisible focused bands. The separated bands of the pI markers (containing separated proteins) were excised, and the pI markers were eluted from each gel piece by water/ethanol and identified by MALDI-TOF/TOF MS. From the washed gel pieces the remaining carrier ampholytes were then washed out and proteins were in-gel digested with trypsin. The obtained peptides were measured by MALDI-TOF/TOF MS and the proteins identified via a protein database search. This procedure allows avoiding time-consuming protein staining and destaining procedures, which shortens the analysis time roughly by half. For comparison, IEF gels were stained with Coomassie Brilliant Blue R 250 and proteins in the gel bands were identified according to the standard proteomic protocol. This work has confirmed that our approach can give information about the correct pI values of particular proteins and shorten significantly the time of analysis.     

Protein profiling by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

An automated system for intact protein analysis is described that combines capillary isoelectric focusing (CIEF), reversed-phase liquid chromatography (RPLC), and electrospray ionization-mass spectrometry (ESI-MS). Performance is demonstrated with a complex yeast enzyme concentrate. CIEF is performed with a microdialysis membrane-based cathodic cell that permits pI fractions to be sampled and stored for subsequent LC-MS analysis. A total of 50 microg protein is loaded onto the capillary. Ten fractions are stored which span the pI range 3-10. Each fraction is subsequently cleaned on a reversed-phase trap column and then characterized by LC-MS. MaxEnt1 is used to deconvolute the raw mass spectra to obtain the molecular weight (MW) of intact proteins/peptides in the sample. A two-dimensional display of pI vs. MW is illustrated for the 500 most prevalent species as identified by MaxEnt1.     

Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations

Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains.     

Speciation and subcellular location of Se-containing proteins in human liver studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and hydride generation-atomic fluorescence spectrometric detection

Speciation of Se-containing proteins in the subcellular fractions of human liver was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by hydride generation-atomic fluorescence spectrometric (HG-AFS) detection. It was found that about 24 kinds of Se-containing proteins existed in subcellular fractions of normal human liver. The molecular weights (MW) of the subunits were mostly in the range 20-30 kDa and 50-80 kDa. Major Se-containing protein fractions at 61 kDa and 21 kDa are probably selenoprotein P and glutathione peroxidase, respectively. The 54 kDa protein is probably a thioredoxin reductase, which is presented in nuclei, mitochondria, lysosome, microsome and cytosol. We noticed that the Se-containing protein with the lowest MW of 9.3 kDa only existed in lysosome. Most of the proteins have not been identified and would require further investigation to characterize them. The specific subcellular distributions of different Se-containing proteins suggest that they could play important biological roles in each organelle.     

Thin-layer agarose isoelectric focusing of alkaline phosphatase isoenzymes from human neutrophils

An improved method for thin-layer agarose isoelectric focusing of alkaline phosphatases (AP) from human neutrophils is described. The solubilization of AP isoenzymes was studied with four detergents. The best results were obtained after sonication with Zwittergent 3-12 (1% final concentration), followed by butanol extraction and ultracentrifugation 105,000 x g for 1 h. The cytosol can be stored at 0 degrees C or -80 degrees C, but not at -20 degrees C. Dialysis of the cytosol against a Tris buffer, pH 7.5, was imperative prior to focusing for removal of the detergent. Enzyme visualization is enhanced by incorporation of ZnCl2 (3 mM) into the agarose gel. The focusing patterns consist of two sets of isoenzymes: two main zones with pI 6.4 and 6.8 and minor components with pI 4.2, 4.8 and 5.2.     

Isoelectric focusing studies of concanavalin A and the lentil lectin

Isoelectric focusing (IEF) of metallized and demetallized preparations of concanavalin A (Con A) consisting of either intact or fragmented subunits shows different band patterns. Metallized Con A consisting of intact polypeptide chains (intact Con A) has an isoelectric point (pI) 8.35. Metallized preparations consisting of fragmented chains (fragmented Con A) show three bands with pI values 8.0, 7.8 and 7.7. Demetallized intact Con A (intact apoCon A) has a pI of 6.5, however, it undergoes pH dependent association during IEF under certain conditions, which gives rise to multiple bands. Ampholyte-mediated demetallization of intact and fragmented Con A and subsequent aggregation of the apoprotein results in multiple bands during IEF in the presence of the pH range 3 to 10 ampholytes. However, ampholytes of the pH range 7 to 9 do not demetallize the proteins and show a single band with intact Con A. The pI of intact Con A remains essentially the same in the presence of inhibitory sugar. Furthermore, different moleculars forms of Con A, including locked and unlocked conformers of intact apoCon A, and the dimeric and tetramic states of both intact Con A and intact apoCon A have been identified and their pI values determined. IEF of the lentil isoelectins, LcH-A and LcH-B, shows single bands of pI 8.5 and 9.0, respectively. However, the native lectin mixture gives rise to an additional band of pI 8.8 due to a hybrid protein formed by ampholyte-mediated subunit exchange between the isolectins.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Colored, low molecular weight pI markers have been developed for isoelectric focusing (IEF) in acidic pH range. Their isoelectric points (pIs) were determined by direct measurement of the pH of the focused bands after completion of IEF on polyacrylamide gels. The practicable suitability of the proposed pI markers as pI standards for IEF was tested by applying gel IEF. The acidic pH gradient was created either by commercial synthetic carrier ampholytes or by mixture of simple buffers consisting of acids (non-ampholytes) and ampholytic buffers. By applying simple acids, it was possible to extend the acidic pH range beyond those achievable with commercial synthetic carrier ampholytes. By using an experimental arrangement without electrode electrolyte reservoirs with electrodes creating the fixed end of the gel, the strongly acidic pH gradient was stable even for prolonged focusing time.     

Capillary isoelectric focusing with UV-induced fluorescence detection

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.     

The use of immobilized pH gradients for the detection of human polymorphisms in the forensic identification of bloodstains.

Some polymorphic proteins (alpha 1-antitrypsin, orosomucoid, transferrin, group specific component, plasminogen) and enzymes (phosphoglucomutase, acid phosphatase, estrase D) were determined in bloodstain extracts by isoelectric focusing with carrier ampholytes (CA) and with immobilized pH gradients (IPGs) rehydrated with CA. IPGs yield superior results for typing of genetics markers in bloodstains since phenotypes are better distinguished and the bands are straighter and sharper in the presence of contaminants. Also, the sensitivity of IPGs with CA is similar to isoelectric focusing (IEF) with CA. A new variant, ACP*B1, found in Negroid west African populations and not found in Caucasians is described. Such a variant can only be determined by IPGs since its isoelectric point (pI 5.95) is close to that of the ACP*B (pI 6.05) variant.     

Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixture

The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3相似文献   

Analysis of metal-containing proteins by gel electrophoresis and synchrotron radiation X-ray fluorescence

For the analysis of metal-containing proteins, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been combined with synchrotron radiation X-ray fluorescence (SRXRF). In a pilot study the applicability of this combined method was tested in the analysis of metalloaded apoazurin and of the selenoproteins in rat testis homogenate. It was shown that it can be applied in the determination of the major stable binding forms of trace elements. After further improvement of the limits of detection the method will allow the analysis of trace element-containing proteins present in the samples at low concentrations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Extension of separation range in capillary isoelectric focusing for resolving highly basic biomolecules

The non-availability of commercial carrier ampholytes in the pH range greater than 11 has contributed to difficulties in focusing and resolving highly basic proteins/peptides using capillary isoelectric focusing (cIEF). Two different approaches, involving the use of N,N,N',N'-tetramethylethylenediamine (TEMED) and ampholyte 9-11, are investigated for their effects on the extension of separation range in cIEF. The addition of TEMED into pharmalyte 3-10 not only prevents the peptides/proteins from focusing in sections of the capillary beyond the detection point, but also extends the separation range to at least isoelectric point (pI) 12. The combination of ampholyte 9-11 with pharmalyte 3-10 surprisingly provides baseline resolution between bradykinin (pI 12) and cytochrome c (pI 10.3). The sample mixture, containing bradykinin, the high-pI protein calibration kit (pI 5.2-10.3), and cytochrome c digest, is employed to demonstrate the cIEF separation of proteins and peptides over a wide pH range of 3.7-12.     

Preparative free-flow isoelectric focusing: modeling and experiments

Free-flow isoelectric focusing was adapted to preparative scale separations and chemical engineering methods were used to describe the main mechanisms operating in the apparatus. A mixture of human serum albumin (pI 4.6) and beta-lactoglobulin (pI 5.22) was separated in pH gradients, generated with carrier ampholytes of different origin and covering the pH ranges 4-6.5, 3.5-5, 4-5.5 and 4.5-5.0. Best results were obtained in the pH 4-5.5 range. The experimental results have validated the results obtained with a numerical model.     

Fluorescein-based pI markers for capillary isoelectric focusing with laser-induced fluorescence detection

We prepared a series of low-molecular-mass fluorescent ampholytes with narrow pI range. These fluorescein-based ampholytes are detection compatible with argon laser-induced fluorescence (LIF) detection. The selected properties, important for their routine use as fluorescent pI markers, were examined. The pI values of new fluorescein-based pI markers were determined by capillary isoelectric focusing (CIEF) using currently available low-molecular-mass pI markers for CIEF with photometric detection. The examples of CIEF with fluorometric detection of new compounds together with fluorescein isothiocyanate (FITC) derivatized proteins are presented.     

高效液相色谱-同位素稀释-电感耦合等离子体质谱法研究大鼠体内的汞结合蛋白

利用高效液相色谱(HPLC)和电感耦合等离子体质谱(ICP-MS)联用技术,对经胚胎早期甲基汞暴露的母、仔鼠脑胞液和肝胞液中汞结合蛋白进行了研究。采用柱后(Post-Column)同位素稀释法(ID)技术定量计算了母、仔鼠组织胞液中与蛋白结合的汞。HPLC-ID-ICP-MS方法的汞回收率与直接ICP-IDMS法的实验结果一致。该方法可以克服生物样品微量元素化合物分析中灵敏度低和标准蛋白不足的缺点,有利于生物体内低丰度金属结合蛋白的分离分析和定量研究。