The element sulfur is almost omnipresent in all natural proteomes and plays a key role in protein quantification. Incorporated
in the amino acids cysteine and methionine, it has been served as target for many protein-labeling reactions in classic quantitative
proteomic approaches based on electrospray or MALDI mass spectrometry. This critical review discusses the potential and limitations
of sulfur isotope dilution analysis (IDA) by inductively coupled plasma—mass spectrometry (ICP-MS) for absolute protein quantification.
The development of this approach was made possible due to the improved sensitivity and accuracy of sulfur isotope ratio measurement
by ICP-MS in recent years. The unique feature of ICP-MS, compound-independent ionization, enables compound (species)-unspecific
sulfur IDA. This has the main advantage that only one generic sulfur standard (i.e., one isotopically labeled sulfur spike)
is required to quantify each peptide or protein in a sample provided that they are completely separated in chromatography
or electrophoresis and that their identities are known. The principles of this approach are illustrated with selected examples
from the literature. The discussion includes also related fields of P/S and metal/S ratio measurements for the determination
of phosphorylation degrees of proteins and stoichiometries in metalloproteins, respectively. Emerging new areas and future
trends such as protein derivatization with metal tags for improved sensitivity of protein detection in ICP-MS are discussed.
Figure The key role of sulfur in protein quantification 相似文献
Nano- and/or macrostructuring of electrode surfaces has recently emerged as a powerful method of improving the performances
of electrochemical devices by enhancing both molecular accessibility and rapid mass transport via diffusion, by increasing
the electroactive surface area in comparison to the geometric one, and/or by providing confinement platforms for hosting suitable
reagents. This brief overview highlights how template technology offers advantages in terms of designing new types of porous
electrodes—mostly based on thin films, and functionalized or not—and discusses their use in analytical chemistry via some
recent examples from the literature on electrochemical sensors and biosensors.
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A molecular iodine selective membrane has been used for preconcentration of I2 generated in situ by iodometric reaction of with excess I− in acidic medium (pH 1–2). This iodometric reaction amplifies the iodine content six times resulting in enhancement of analytical
response ranging from three times for molecular methods to six times for elemental methods. The chemical conditions of this
iodometric reaction were optimized for quantitative generation and subsequent sorption of I2 in the membrane samples (96 ± 3%). The homogeneous transparent membrane was prepared by immobilizing I2-complexing polyvinylpyrrolidone (PVP) in the plasticized cellulose triacetate matrix. Four different analytical methods were
examined for quantitative determination of in iodized salt samples by preconcentrating it as I2 in the membrane matrix. These methods were: (1) spectrophotometry of the PVP-I2 complex formed in the membrane matrix, (2) a radiotracer method using I− tagged with 131I radiotracer, (3) instrumental neutron activation analysis (INAA), and (4) energy-dispersive X-ray fluorescence (EDXRF) analysis.
The contents thus determined in the iodized salt samples by the membrane-based radiotracer method were compared with the total
iodine determined in salt samples by epithermal instrumental neutron activation analysis (EINAA). The membrane-based method
for iodate determination in salt samples has advantages over conventional analytical methods, for example preconcentration
and chemical amplification, and is free from interference from anions.
Figure A molecular iodine selective membrane was used for the quantitative preconcentration of I2 generated in situ by iodometric reaction of with excess Iaˆ’ in acidic medium, which amplifies iodine content six times 相似文献
This paper describes the situation that can emerge when the signals to be evaluated in quantitative NMR measurements—so-called “monitor signals”—consist of several resonance lines from the stereoisomers of the analyte in addition to an impurity signal underneath. The monitor signal problem is demonstrated in the purity assessment of two samples of 2-(isopropylamino)-4-(ethylamino)-6-chloro-1,3,5-triazine (atrazine), a common herbizide which served as analyte in a CCQM intercomparison. It is shown that, in DMSO-d6 solution, a mixture of stereoisomers leads to several individual overlapping singlets, which are further split by spin–spin coupling. A measurement protocol was developed for finding and identifying an impurity that has a signal that is positioned precisely beneath the methyl signal chosen as the monitor signal in one of the samples. Quantitative NMR purity assessment is still possible in this special case, but with higher uncertainty.
Electronic Supplementary Material Supplementary material is available for this article at 相似文献
In-torch LA–ICP–MS was implemented into an in-house-built ICP–TOFMS system. The fast data acquisition capabilities of the
new configuration allowed simultaneous multi-element measurement and readout of in-torch LA–ICP–MS signals with 30 μs time
resolution. The measurements confirmed previously observed fine structures of in-torch generated signals and provided new
insights in the dynamic processes in the plasma on a microsecond time scale. The new setup is described in detail and first
figures of merit are given.
Figure Time dependent multi element signal after laser ablation in the torch of an ICP-TOFMS instrument 相似文献
Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach.
An analytical procedure for the simultaneous determination in human plasma and oral fluids of several illicit drugs belonging
to different chemical and toxicological classes is presented. Amphetamine, methamphetamine, morphine, 6-monoacetylmorphine,
methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, cocaine, benzoylecgonine, tetrahydrocannabinol,
carboxytetrahydrocannabinol, ketamine, and phencyclidine have been quantified in real samples using a very rapid sample treatment,
basically a protein precipitation. The quantitative analysis was performed by liquid chromatography–tandem mass spectrometry
and has been fully validated. All the analytes were detected in positive ionization mode using a TurboIonSpray source, except
carboxytetrahydrocannabinol, which was detected in negative ionization mode. The use of a diverter valve between the column
and the mass spectrometer allows the preservation of the ion source performances for high-throughput analysis.
Figure Diverter system 相似文献
In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of
peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional
chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange
chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides.
Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional
separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative
to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational
modifications.
Figure Artistic impression of the HILIC separation mechanism 相似文献
Calcareous stones have been largely used to build historical buildings. Among these, the calcarenites are usually characterized
by a high content of calcite and a high open porosity, which make them very sensitive to the weathering caused by physical
and chemical agents. In order to prevent their deterioration and to retard their decay, different protective products—mainly
polymers—are applied on the stone artefact surfaces. In this work we apply the methodology tested in a preliminary study to
investigate the morphological changes of the internal structure of a biocalcarenite (Lecce stone) by micro x-ray computed
tomography (μ-CT). The porosity and other morphological parameters of the rock before and after the conservation treatment
were calculated on a significant number of samples. The Student’s t test was applied for statistical comparison. The results reveal that the treatment with Paraloid B72 (PB 72) is homogenously
distributed and causes small changes to the natural properties of the rock, whereas the application of a fluoroelastomer (NH)
causes an appreciable decrease in porosity and variation in terms of wall thickness distribution, probably resulting from
its inhomogeneous distribution.
Figure Porosity and other morphological parameters of Lecce stone were investigated by μ-CT: the effect of conservation treatment
with fluoroelastomer on wall thickness distribution is illustrated 相似文献
Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC–MS a procedure
to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation
of bound and unbound marker—the most crucial step of the assay—could be effectively achieved by filtration in a 96-well-format.
MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence
of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using
1 mol L−1 NaCl in the incubation milieu reveals remarkable differences with respect to the marker’s affinity and kinetics and to the
investigated test compound’s potency.
Principle of MS binding assays After incubation of a target with a native marker, bound and unbound marker are separated by filtration. Subsequently, the
bound native marker is liberated from the target and finally quantified by LC-MS-MS.
Dedicated to Prof. Hans-Dietrich Stachel on the occasion of his 80th birthday 相似文献
The membrane proteomes of a wild-type Corynebacterium glutamicum and an L-lysine-producing strain were quantitatively analyzed by two complementary proteomics techniques—anion exchange chromatography
AIEC/SDS-PAGE and 16BAC-PAGE/SDS-PAGE—and the results were compared. Although both techniques allow for the fast screening
of differences in protein abundance, AIEC/SDS-PAGE was superior to 16BAC-PAGE/SDS-PAGE with respect to protein separation,
it was more suitable for relative protein quantification, and allowed more differentially regulated proteins to be detected
(the succinate dehydrogenase complex, an ABC-type cobalamin/Fe3+ siderophore transport system, the maltose binding protein, and a subunit of the cytochrome bc-aa3 supercomplex were upregulated, while a periplasmic component of an ABC-type transporter and an iron-regulated ABC-type transporter
were downregulated in the producer). The results indicate the important role of tricarboxylic acid cycle enzymes as well as
the adaptation of transport processes in L-lysine-producing cells. Since the only genetic differences between the wild type and the L-lysine producer occur between four central metabolic enzymes in the cytoplasm, our study illustrates the complex effects
of metabolic engineering on cell physiology and the power of the new AIEC/SDS-PAGE proteomics approach to detect these effects.
相似文献
In the present work we report the results obtained with a methodology based on direct coupling of a headspace generator to
a mass spectrometer for the identification of different types of petroleum crudes in polluted soils. With no prior treatment,
the samples are subjected to the headspace generation process and the volatiles generated are introduced directly into the
mass spectrometer, thereby obtaining a fingerprint of volatiles in the sample analysed. The mass spectrum corresponding to
the mass/charge ratios (m/z) contains the information related to the composition of the headspace and is used as the analytical signal for the characterization
of the samples. The signals obtained for the different samples were treated by chemometric techniques to obtain the desired
information. The main advantage of the proposed methodology is that no prior chromatographic separation and no sample manipulation
are required. The method is rapid, simple and, in view of the results, highly promising for the implementation of a new approach
for oil spill identification in soils.
Figure PCA score plots illustrate clear discrimination of types of crude oil in polluted soil samples (e.g. results are shown for
vertisol) 相似文献
Archaeological samples originating from a cemetery of a Roman settlement, Pretorium Agrippinae (1st–3rd century A.D.), excavated near Valkenburg (The Netherlands) have been subjected to Pb isotopic analysis. The set
of samples analysed consisted of infant bone tissue and possible sources of bone lead, such as the surrounding soil, garum,
and lead objects (e.g., water pipes). After sample digestion with quantitative Pb recovery and subsequent quantitative and
pure isolation of lead, the Pb isotopic composition was determined via multicollector ICP–mass spectrometry. The Pb isotope
ratio results allowed distinction of three groups: bone, soil, and lead objects + garum. The 208Pb/206Pb ratio ranges were between 2.059 and 2.081 for the soils, between 2.067 and 2.085 for the bones, and between 2.087 and 2.088
for the lead objects. The garum sample is characterised by a 208Pb/206Pb ratio of 2.085. The bone group is situated on the mixing line between the soil and lead object groups, allowing the statement
that diagenesis is not the main cause of the Pb found in the bones.
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A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for
the simultaneous determination of sulfamonomethoxine and its metabolites, N4-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation
were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml−1, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all
and is, therefore, harmless to the environment and humans.
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The standard methods currently used to read out microarrays are fluorescent and chemiluminesent imaging techniques. These
methods require labeling of a component with a marker and, usually, only the concentration of the marker molecule is detected.
A label-free imaging method that also enables quantitative spectroscopic analysis of the composition and component interaction
would be of great advantage. In this article it is shown for the first time that IR mapping ellipsometry enables label-free
imaging of a biochip before and after incubation with peptide solution. The measurements prove that IR ellipsometry is a sensitive
tool for laterally resolved identification of the different materials and determination of the composition of a biochip. The
lateral resolution required was achieved by using radiation from an infrared synchrotron beamline.
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A chemical prototype sensor was constructed based on nanofiber-structured TiO2 and highly sensitive quartz resonators. The gas-sensing behavior of this new sensor to selected simulant warfare agents was
investigated at room temperature. Results showed rapid response and good reversibility of this sensor when used with high-purity
nitrogen. This provides a simple approach to preparation of materials needed as chemical sensors for selected organic volatiles
or warfare agents.
Figure Sensing behavior of TiO2 nanofiber sensor to chemical vapors 相似文献
Abstract The enzymatic cyclization of phenoxyacetylcyanomethylenetriphenylphosphorane derivatives using pre-treated baker’s yeast cells,
a rapid one-pot assembly of heteromacrocyclic compounds, is described.
Graphical abstract
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Figure Schematic diagram of a typical arrangement used for hyphenating chemical microseparations (e.g. capillary HPLC, CE, or CEC)
with microcoil NMR detection 相似文献
The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration
(GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this
study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software
has been developed for IGF-I to facilitate its quantification by HPLC–electrospray–ion-trap mass spectrometry. Second, product-ion
scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma samples, fortified plasma samples,
and equine GH post-administration samples. This “top-down” approach method enables characterisation of fragment ions corresponding
to the carboxy terminal end, which can be useful for the confirmation of the presence of IGF-I in plasma samples.
Figure Structure of IGF-I and amino acid sequences of IGF-I and R3 IGF-I. Deconvolution mass spectra of the IGF-I and R3 IGF-I mixture 相似文献
A series of octadecylsilane-modified silicas were prepared by sol-gel and grafting methods. Carbon contents and octadecyl
chain conformations were shown to depend on the preparative route. Grafting engenders a low carbon content and a liquid-like
chain conformation, while the sol-gel method affords a much higher carbon content and a crystalline conformation. The relationships
between the toluene adsorption of the hybrid silicas and their chain conformations, their carbon contents and their textural
characteristics are discussed. These sorbents, when used in combination with ultraviolet diffuse reflectance spectroscopy
(UV DRS), can be employed as a rapid screening method for detection of aromatic compounds in water and air environmental matrices.
Figure Octadecylsilane-modified silicas in the adsorption of toluene 相似文献