共查询到20条相似文献,搜索用时 31 毫秒
1.
Ocaña MF Fraser PD Patel RK Halket JM Bramley PM 《Rapid communications in mass spectrometry : RCM》2007,21(3):319-328
The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food-labeling requirements. 相似文献
2.
Mª Concepción García López Virginia Garcia-Cañas Mª Luisa Marina Alegre 《Journal of chromatography. A》2009,1216(43):7222-7228
A reversed-phase high-performance liquid chromatography–electrospray mass spectrometry (RP-HPLC–ESI-MS (ion trap)) method is developed, for the first time, for profiling transgenic and non-transgenic maize with the aim of cultivar characterization. To optimize chromatographic conditions the following parameters were studied: column, gradient, and ion-pairing reagent. Moreover, the influence in the MS signal of the variation of the capillary voltage and the accumulated ions in the trap was also studied. The developed method was applied to the profiling of different protein fractions (albumin, globulin, prolamin, and glutelin) isolated from Bt transgenic and non-transgenic maize cultivars. Moreover, different maize samples, namely, maize cultivars from different geographical origins (USA, Canada, France, and Spain), transgenic maize samples with certified GMO content, and three transgenic Bt maize cultivars with their isogenic non-transgenic counterparts (Aristis Bt vs. Aristis, PR33P67 vs. PR33P66, and DKC6575 vs. Tietar) were profiled by the developed method. Mass spectra obtained for certain peaks in the maize cultivars studied resulted, in some occasions, useful for cultivar characterization and differentiation. The comparison of UV and MS profiles and mass spectra corresponding to the protein fractions with those of the whole seeds enabled the assignment of some peaks. 相似文献
3.
Stephen A. Wise Dianne L. Poster Stefan D. Leigh Catherine A. Rimmer Stephanie Mössner Patricia Schubert Lane C. Sander Michele M. Schantz 《Analytical and bioanalytical chemistry》2010,398(2):717-728
SRM 1597 Complex Mixture of Polycyclic Aromatic Hydrocarbons from Coal Tar, originally issued in 1987, was recently reanalyzed
and reissued as SRM 1597a with 34 certified, 46 reference, and 12 information concentrations (as mass fractions) for polycyclic
aromatic hydrocarbons (PAHs) and polycyclic aromatic sulfur heterocycles (PASHs) including methyl-substituted PAHs and PASHs.
The certified and reference concentrations (as mass fractions) were based on results of analyses of the coal tar material
using multiple analytical techniques including gas chromatography/mass spectrometry on four different stationary phases and
reversed-phase liquid chromatography. SRM 1597a is currently the most extensively characterized SRM for PAHs and PASHs. 相似文献
4.
Size‐exclusion liquid chromatography was coupled to UV and inductively coupled plasma mass spectrometry (ICP‐MS) for detection to perform elemental speciation studies on different edible mushrooms. Molecular weight (MW) distribution patterns of several elements among different fractions present in various edible mushrooms are presented. The association of the elements with the high and low MW fractions was observed using sequential detection by UV and ICP‐MS. Separation was performed using a Superdex 75 column. Variability of the fractionation patterns with three different extraction media (0.05 mol l?1 NaOH; 0.05 mol l?1 HCl; hot water at 60°C) was evaluated for mushroom species. A comparative elemental speciation study was performed in order to determine the differences in the fractionation patterns of silver, arsenic, cadmium, mercury, lead, and tin in Boletus edulis, Agaricus bisporus, and Lentinus edodes. Differences in the fractionation patterns of the elements were found to depend on the mushroom species and the extraction medium. Most of the elements were associated with high mw fractions. It was not possible to assess the trace metal contributions from the mushroom growth media. Copyright © 2004 John Wiley & Sons, Ltd. 相似文献
5.
Gergely V Kubachka KM Mounicou S Fodor P Caruso JA 《Journal of chromatography. A》2006,1101(1-2):94-102
In this study, selenium species from Se containing proteins in mushrooms (Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1M NaOH, 30 mM Tris-HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris-HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample. 相似文献
6.
Koomen JM Zhao H Li D Nasser W Hawke DH Abbruzzese JL Baggerly KA Kobayashi R 《Rapid communications in mass spectrometry : RCM》2005,19(12):1624-1636
A peptide targeting method has been developed for diagnostic protein discovery, which combines proteolytic digestion of fractionated plasma proteins and liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) profiling. Proteolysis prior to profiling overcomes molecular weight limitations and compensates for the poor sensitivity of matrix-assisted laser desorption/ionization (MALDI) protein profiling. LC/MS increases the peak capacity compared to crude fractionation techniques or single sample MALDI analysis. Differentially expressed peptides are targeted in the mass chromatograms using bioinformatic techniques and subsequently sequenced with MALDI tandem MS. In a model study comparing pancreatic cancer patients to controls, 74% of the peptide targets were successfully sequenced. This profiling method was superior to previous experiments using single sample MALDI analysis for protein profiling or proteolytic peptide profiling, because more potential protein markers were identified. 相似文献
7.
Molecular size distribution patterns of Cu, Mn, Ni, and Zn were determined in several nut species by size-exclusion liquid chromatography (SEC) coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP–MS) for detection. The molecular weight (MW) fractionation of the different metals was performed with a Superdex Peptide column, injecting 100 L of the extracted solutions. The association of the elements with different MW fractions was observed with sequential detection by UV and ICP–MS. Various separation conditions were evaluated to obtain proper resolution and reproducible results with the size-exclusion column. Complete MW information of the elemental fractions in the nut samples was obtained within a retention time of 30 min. Fractionation of the above mentioned elements was done in nine different nut species commonly found in commercial markets. Variability of the fractionation patterns for two different extraction media, 0.05 mol L–1 NaOH and 0.05 mol L–1 HCl, was evaluated for every nut sample. Differences in the elemental fractionation patterns were found depending on the extraction procedure, nut species, and the type of element studied. It was also observed that the elements studied showed predominant association with high MW fractions when extracted with basic solution whereas with acidic extraction media only low MW fractions were obtained. 相似文献
8.
Alfredo Sanz-Medel Ana B Soldado CabezueloRadmila Mila?i? Tjasa Bantan Polak 《Coordination chemistry reviews》2002,228(2):373-383
Chemical speciation of aluminium in the low molecular mass (LMM) and high molecular mass (HMM) fractions of human serum is discussed. A critical review of the literature on different analytical procedures described for the speciation of aluminium in human serum samples is presented here. The methodologies, the experimental and instrumental requirements and the ability of the reported analytical procedures for identification of HMM and LMM aluminium species in human serum are examined in detail. Non-chromatographic separations coupled to electrothermal atomic absorption spectrometry for aluminium detection are compared with chromatographic techniques (size exclusion chromatography, anion exchange chromatography and fast protein liquid chromatography) coupled to ETAAS or inductively coupled plasma mass spectrometry (ICP-MS) detection for Al-HMM species investigations. Studies and techniques reported for Al-LMM compounds are also summarised, both for healthy volunteers and dialysis patients. On the basis of the knowledge obtained from the application of the developed analytical procedures to real serum analysis, it has been demonstrated that most of Al in human serum is bound to Al-transferrin, while the LMM-Al fraction (10-20% of total Al) mainly contains Al-citrate, Al-phosphate and ternary Al-citrate-phosphate complexes. 相似文献
9.
A method is described for the determination of three- to six-ring polynuclear aromatic hydro-carbons (PAHs) and mononitrated PAHs (nitro-PAHs) in particulate matter. The procedure includes Soxhlet extraction followed by fractionation using gel filtration chromatography and normal-phase liquid chromatography with an aminosilane stationary phase. The resulting fractions were separated by capillary gas chromatography (GC) into individual PAHs and nitro-PAHs which were detected by a flame-ionization detector and a thermal energy analyzer, respectively. Commercially available standards were used for quantification. Individual peak assignments were confirmed by using both mass spectral and retention index data obtained through computerized capillary GC-mass spectrometry. Several samples were processed, including a certified diesel particulate reference material supplied by the National Bureau of Standards for the purpose of evaluating analytical methods. This method may also be applicable in the determination of certain carbonyl PAHs. 相似文献
10.
Shikov AN Ossipov VI Martiskainen O Pozharitskaya ON Ivanova SA Makarov VG 《Journal of chromatography. A》2011,1218(50):9111-9114
Thin-layer chromatography (TLC) with off-line high-performance liquid chromatography coupled to diode array detection and micrOTOF-Q mass spectrometry (HPLC-DAD-MS) resulted in the successful fractionation, separation and identification of spinochrome pigments from sea urchin (Strongylocentrotus droebachiensis) shells. Two fractions of pigments were separated by TLC and eluted with methanol using a TLC-MS interface. HPLC-DAD-MS analysis of the fractions indicated the presence of six sea urchin pigments: spinochrome monomers B and D, three spinochrome dimers (anhydroethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin) and its isomer and ethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin)), and one pigment that was preliminary identified as a spinochrome dimer with the structural formula C(22)H(16)O(16). 相似文献
11.
Wang Y Hancock WS Weber G Eckerskorn C Palmer-Toy D 《Journal of chromatography. A》2004,1053(1-2):269-278
The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step. In this paper, we explored the advantages of a combination of FFE and liquid chromatography/mass spectrometry to extend the dynamic range of a proteomic analysis of a complex cell lysate. In this study, the whole cell lysate of a chronic myelogeneous leukemia cell line, K562/CR3, was prefractionated by FFE into 96 fractions spanning pH 3-12. Of these, 35 fractions were digested with trypsin and then analyzed by LC/MS. Depending on the algorithm used for peptide assignment from MS/MS data, at least 319 proteins were identified through database searches. The results also suggested that pI could serve as an additional criterion besides peptide fragmentation pattern for protein identification, although in some cases, a pI shift might indicate post-translational modification. In summary, this study demonstrated that free flow electrophoresis provided a useful prefractionation step for proteomic analysis and when combined with LC/MS allowed the identification of significant number of low level proteins in complex samples. 相似文献
12.
Feng B Patel AH Keller PM Slemmon JR 《Rapid communications in mass spectrometry : RCM》2001,15(10):821-826
The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-containing fractions from column chromatography in less than 1 hour using parallel LC/MS. 相似文献
13.
Lattanzio VM Solfrizzo M Powers S Visconti A 《Rapid communications in mass spectrometry : RCM》2007,21(20):3253-3261
A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%. 相似文献
14.
Flow field-flow fractionation (FlFFF) has been utilized for size-based separation of rat liver mitochondria. Collected fractions of mitochondria of various sizes were examined by confocal microscopy, and mitochondria of each fraction were lysed and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the comparison of protein patterns in differently sized mitochondria by densitometric measurements, and for protein characterization of some gel spots with nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). FlFFF fractions of the mitochondria were also tryptically digested for shotgun proteomic characterization of mitochondrial proteins/peptides by nLC-ESI-MS-MS. Peak area (integrated ion counts) of some peptides extracted from LC-MS chromatograms were examined at different fractions for the quantitative comparison. Among 130 proteins, 105 unique proteins were found to be mitochodrial from the off-line combination of FlFFF and nLC-ESI-MS-MS analysis. It also showed that 23 proteins were found in all fractions but some proteins were found exclusively in certain fractions. Among 25 proteins listed from other subcellular species, seven proteins were known to exist in mitochondria as well as in other subcellular locations, which may support the possible translocation or multiple localizations of proteins among organelles. This study demonstrated effective use of FlFFF for the isolation and/or enrichment of intact mitochondria isolated from cells, as well as its potential use for the fractionation of other subcellular components in the framework of subcellular functional proteomics. 相似文献
15.
Yanes O Avilés FX Wenzel R Nazabal A Zenobi R Calvete JJ 《Journal of the American Society for Mass Spectrometry》2007,18(4):600-606
Proteomic profiling involves identification and quantification of protein components in complex biological systems. Most of the mass profiling studies performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been restricted to peptides and small proteins (<20 kDa) because the sensitivity of the standard ion detectors decreases with increasing ion mass. Here we perform a protein profiling study of the snake venom Sistrurus miliarius barbouri, comparing 2D gel electrophoresis and reversed-phase high-performance liquid chromatography (HPLC) with a high mass cryodetector MALDI-TOF instrument (Macromizer), whose detector displays an uniform sensitivity with mass. Our results show that such MS approach can render superior analysis of protein complexity compared with that obtained with the electrophoretic and chromatographic approaches. The summation of ion impacts allows relative quantification of different proteins, and the number of ion counts correlates with the peak areas in the reversed-phase HPLC. Furthermore, the sensitivity reached with the high mass cryodetection MS technology clearly exceeds the detection limit of standard high-sensitivity staining methods. 相似文献
16.
The complexity of the human serum proteome is attributed to both a large dynamic range of protein abundance, as much as 10 orders of magnitude, and a disproportionate few dozens of proteins representing as much as 99% of the total protein content. These characteristics make it beneficial to use a pre-fractionation step prior to any high-resolution analysis, such as mass spectrometry. The present method describes a unimodal multidimensional chromatography concept to rapidly achieve an effective fractionation of human serum that is directly amenable with surface-enhanced laser desorption/ionization (SELDI)-based mass spectrometry. This method is based on the use of a column composed of a superimposed sequence of sorbents. The assembly is first equilibrated with a single binding buffer and then loaded with the whole crude sample. As the sample crosses the different adsorbent layers proteins within are sequentially trapped according to the complementary properties vis-a-vis of the sorbent. Once the loading and capturing is achieved, the sequence of columns is disassembled and each column, containing different complement of proteins is eluted separately in a single step and under optimal elution conditions. When compared to classical single-chemistry fractionation based on, for example, anion-exchange and pH stepwise elution, the new proposed approach shows much lower protein overlap between fractions, and therefore, greater resolution. This results in a larger number of detectable species, and therefore, reinforces the power of discovery of new biomarkers. A significantly higher sensitivity for low-abundance species was additionally found as evidenced by spiking trials. 相似文献
17.
Our previous study found that high-molecular-weight selenium (Se) species make up 82% of the total Se in the bean of Se-enriched
soybean plants (Chan et al. 2010, Metallomics, 2(2): p. 147–153). The Se species have been commonly seen in other plants in addition to soybean, but their identities remain
unresolved. The present study employs a multi-technique metallomics approach to characterize the proteins containing Se in
the beans of Se-enriched soybean plants. Two main categories of proteins, maturation proteins and protease inhibitors, were
found in Se-containing high-performance liquid chromatography (HPLC) fractions. The proteins were screened by two-dimensional
HPLC-inductively coupled plasma mass spectrometry, size-exclusion chromatography, and anion-exchange chromatography, and the
Se-containing fractions were then identified by peptide mapping using HPLC-Chip-electrospray ion trap mass spectrometry. Based
on the belief that Se goes into proteins through non-specific incorporation, a new method was designed and applied for the
Se-containing peptide identification. The Se-containing peptide KSDQSSSYDDDEYSKPCCDLCMCTRS, part of the sequence of protein
Bowman–Birk proteinase isoinhibitor (Glycine max), was found in one of the Se-containing fractions. The nutritional value of the Se-containing proteins in Se-enriched soybeans
will be an interesting topic for the future studies. 相似文献
18.
不同石斛枫斗中酚酸类活性成分的比较及杓唇石斛素和石斛酚含量的测定 总被引:1,自引:0,他引:1
建立了基于液相色谱-离子阱飞行时间串联质谱(LC-IT-TOF MS/MS)的酚酸类分析方法,对6种共18批次的石斛枫斗药材中的酚类化合物进行了研究,其中18种酚酸类化合物得到初步定性。在共有化合物中选择杓唇石斛素和石斛酚作为质量控制的目标化合物。在此基础上,采用响应面法(RSM)确定了提取溶剂为60%甲醇、提取时间为65 min的超声提取杓唇石斛素和石斛酚的最佳条件,建立了石斛枫斗中杓唇石斛素、石斛酚的高效液相色谱(HPLC)含量测定方法。定量线性优于0.999 8,检出限(LOD)分别为0.18 mg/L和0.09 mg/L,重复性的相对标准偏差(RSD)小于3%,加标回收率均值分别为97.1%和101.4%。这些结果表明本研究建立的方法可用于石斛枫斗药材中酚酸类化学成分研究及质量控制。 相似文献
19.
沙特常压渣油超临界流体萃取分馏宽馏分中含硫化合物的分子表征 《燃料化学学报》2016,44(2):177-183
采用超临界流体萃取分馏(SFEF)装置,将沙特中质原油常压渣油(SZAR)按照分子大小和极性强弱进行多级萃取分馏,得到四个宽馏分和一个萃余残渣。考察了四个宽馏分沸程分布、密度、残炭值、黏度、相对分子质量、硫和氮含量以及结构族组成等基本性质均随萃取压力的变化趋势。采用甲基衍生化作用使含硫化合物转化为含有极性的甲基硫盐,结合正离子模式下的电喷雾(ESI)电离源的傅里叶变换离子回旋共振质谱(FT-ICR MS)分析各馏分中所包含的含硫化合物的分子组成。结果表明,超临界萃取分馏技术可以将馏分按照分子大小进行分离,且对于非烃类化合物具有富集作用。各个馏分中含量最高的均为包含一个硫原子的S1类化合物,且二苯并噻吩(DBE=6)类含量最高。馏分越重,S1、S2类化合物含量越高、种类越繁多、且缩合程度更高,包含的更多的大分子含硫化合物。 相似文献
20.
Six fractions containing tri- to pentaglycosylceramides were isolated from the green, fresh water alga Chlorella kessleri, grown heterotrophically, by using preparative high-performance liquid chromatography (HPLC). Up to twelve fractions were obtained by further reversed-phase HPLC of each glycosylceramide. The use of a polar capillary column with Supelcowax 10 as the stationary phase allowed an excellent separation of the individual molecular species of ceramides, even though the separation did not occur when the ceramides differed only in the position of the amide bond. The individual molecular species (even if present in mixtures) were identified by gas chromatography-chemical ionization mass spectrometry. The evidence for a complete structure was obtained by enzyme splitting with alpha- and beta-galactosidases (the sequence of monosaccharides) and by negative ionization fast atom bombardment mass spectrometry. More than 400 molecular species of glycosylceramides were identified. 相似文献