| 1. |
- Ahlford, Annika, et al.
(author)
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Dried reagents for multiplex genotyping by tag-array minisequencing to be used in microfluidic devices
- 2010
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In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 135:9, s. 2377-2385
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Journal article (peer-reviewed)abstract
- We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics.
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| 2. |
- Artemenko, Konstantin A., et al.
(author)
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Optimization of immunoaffinity enrichment and detection : toward a comprehensive characterization of the phosphotyrosine proteome of K562 cells by liquid chromatography-mass spectrometry
- 2011
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In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 136:9, s. 1971-1978
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Journal article (peer-reviewed)abstract
- Phosphorylation of protein tyrosine residues regulates many cell functions and has also been proved to be involved in oncogenesis. Thus, the identification of the phosphotyrosine (pTyr) proteome of cells is a very important task. Since tyrosine phosphorylation represents only around 1% of the total human phosphoproteome, the study of pTyr proteins is rather challenging. Here we report the optimization study of the phosphotyrosine proteome using K562 cells as a model system. A substantial segment of the phosphotyrosine proteome of K562 cells was characterized by immunoaffinity enrichment with 4G10 and PYKD1 antibodies followed by LC-MS/MS analysis. 480 non-redundant pTyr peptides corresponding to 342 pTyr proteins were found. 141 pTyr peptides were not described elsewhere. The mass spectrometry approach involving high-resolving FTMS analysis of precursor ions and subsequent detection of CID fragments in a linear ion trap was considered as optimal. For detection of low abundant pTyr peptides pooling of individual immunoaffinity enrichments for one LC-MS/MS analysis was crucial. The enrichment properties of the monoclonal PYKD1 antibody were presented for the first time, also in comparison to the 4G10 antibody. PYKD1 was found to be more effective for protein enrichment (1.2 and 5% efficiency at peptide and protein level correspondingly), while 4G10 showed better results when peptide enrichment was performed (15% efficiency versus 3.6% at protein level). Substantially different subsets of the phosphoproteome were enriched by these antibodies. This finding together with previous studies demonstrates that comprehensive pTyr proteome characterization by immunoprecipitation requires multiple antibodies to be used for the affinity enrichment.
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| 3. |
- Axelsson, Mikael D., et al.
(author)
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Multielemental analysis of Mn–Fe nodules by ICP-MS: optimisation of analytical method
- 2002
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In: The Analyst. - 0003-2654 .- 1364-5528. ; 127:1, s. 76-82
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Journal article (peer-reviewed)abstract
- Two acid digestion procedures (microwave-assisted and room temperature) were developed for the quantitative analysis of ferromanganese nodules by inductively coupled plasma double focusing sector field mass spectrometry (ICP-SFMS). Different compositions of the acid mixture, dilution factors and corrections for spectral interferences were tested. A combination of nitric, hydrochloric and hydrofluoric acids is necessary for complete sample digestion, with lowest acid to sample ratios (v/m) of 15 and 1.5. respectively, for the last two acids. Sample dilution factors higher than 2 X 104 should be used in order to decrease matrix effects and provide robust long-term instrumental operation. In spite of high dilution. method detection limits in the sub-mug g(-1) range were obtained for 54 out of 71 elements tested. due to the high detection capability of ICP-SFMS, as well as the special care taken to ensure the purity of reagents, to clean the instrument sample introduction system and to minimise sample handling. Owing to the presence of unresolved (at the resolution available) spectral interferences, accurate determination of Au, Hg, Os, Pd, Re and Rh is impossible without matrix separation. The accuracy of the entire analytical method was tested by the analysis of two nodule reference materials. The results generated agreed to within +/-2% for about 10, within +/-10% for more than 40 and within +/-20% for about 50 of 53 elements for which certified, recommended or literature values are available. A precision better than 3%, expressed as the between-digestion relative standard deviation (n=4). was obtained for the majority of elements, except in cases limited by low analyte concentrations
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| 4. |
- Azmi, Jahanara, et al.
(author)
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Metabolic trajectory characterisation of xenobiotic-induced hepatotoxic lesions using statistical batch processing of NMR data : Nicholson Jeremy K., Holmes Elaine
- 2002
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In: Analyst. - : Royal Society of Chemistry (RSC). ; 127, s. 271-6
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Journal article (peer-reviewed)abstract
- Multivariate statistical batch processing (BP) analysis of 1H NMR urine spectra was employed to establish time-dependent metabolic variations in animals treated with the model hepatotoxin, -naphthylisothiocyanate (ANIT). ANIT (100 mg kg-1) was administered orally to rats (n = 5) and urine samples were collected from dosed and matching control rats at time-points up to 168 h post-dose. Urine samples were measured via1H NMR spectroscopy and partial least squares (PLS) based batch processing analysis was used to interpret the spectral data, treating each rat as an individual batch comprising a series of timed urine samples. A model defining the mean urine profile over the 7 day study period was established, together with model confidence limits (±3 standard deviation), for the control group. Samples obtained from ANIT treated animals were evaluated using the control model. Time-dependent deviations from the control model were evident in all ANIT treated animals consisting of glycosuria, bile aciduria, an initial decrease in taurine levels followed by taurinuria and a reduction of tricarboxylic acid cycle intermediate excretion. BP provided an efficient means of visualising the biochemical response to ANIT in terms of both inter-animal variation and net variation in metabolite excretion profiles. BP also allowed multivariate statistical limits for normality to be established and provided a template for defining the sequence of time-dependent metabolic consequences of toxicity in NMR based metabonomic studies.
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| 5. |
- Badea, M, et al.
(author)
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A flow immunoassay for alkylphenol ethoxylate surfactants and their metabolites - questions associated with cross-reactivity, matrix effects, and validation by chromatographic techniques
- 2003
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In: Analyst. - : Royal Society of Chemistry (RSC). - 1364-5528. ; 128:7, s. 849-856
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Journal article (peer-reviewed)abstract
- This paper describes the application and evaluation of a competitive enzyme flow injection immunoassay (EFIIA) for screening of alkylphenol ethoxylate (APEO) surfactants in different water samples based on a generic immunoassay system previously developed ( see E. Burestedt, C Nistor, U. Schagerlof and J. Emneus, Anal. Chem., 2000, 72, 4171 - 4177). The detection limits for octylphenol ethoxylates (OPEOs), nonylphenol ethoxylates (NPEOs), and nonylphenol (NP) were 0.5 mug l(-1), between 2 and 3 mug l(-1), and 50 mug l(-1), respectively, with a sample throughput of 6 h(-1) (i.e., for triplicate analysis of each sample). Different OPEOs and NPEOs were highly cross-reactive within the assay, with sensitivities in the same order of magnitude for all the ethoxylates tested, thus the result obtained by the EFIIA method could be used as an "alkylphenol ethoxylate index". No or minor matrix effects with recoveries between 70 - 120% for the reference analyte NPEO10 in tap, and surface water, and acceptable for rainwater, were observed. Influent and effluent surfactant containing wastewater samples were analysed by EFIIA, LC-MS, LC-Fluoresence (LC-FL), and a commercial microplate ELISA. High recoveries for different concentrations of APEO(10) spiked into a 200 times diluted raw influent and effluent wastewater were achieved with the EFIIA method, however, the found APEO content of the same diluted wastewater samples, before spiking, could not be correlated directly to the chromatographic result by any of the immunoassays, and the possible reasons for this are discussed. The same trend of decreasing APEO content from influent to effluent wastewater could, however, be followed for all methods employed.
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| 6. |
- Bailly-Chouriberry, Ludovic, et al.
(author)
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A new analytical method based on anti-EPO monolith column and LC-FAIMS-MS/MS for the detection of rHuEPOs in horse plasma and urine samples
- 2012
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In: The Analyst. - 0003-2654 .- 1364-5528. ; 137:10, s. 2445-2453
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Journal article (peer-reviewed)abstract
- Recombinant human erythropoietin (rHuEPO) is a 30-34 kDa glycoprotein banned by the racing authorities. For some years this molecule has been detected in race horses in USA and in Europe, and even in racing camels. Although direct methods to differentiate horse endogenous EPO and rHuEPO have been developed either by LC-MS/MS or by isoelectric focusing (IEF) with double-blotting, the short confirmation time of such prohibited hormone in plasma remains a problem for horseracing doping control laboratories. In order to improve the rHuEPOs confirmation process in horse plasma or urine in terms of reliability and delay, a small anti-EPO monolith membrane contained in a disposable column (anti-EPO monolith column) has been successfully used and validated (n = 10). This new sample preparation, combined with LC-FAIMS-MS/MS, has been performed on plasma and urine samples collected from one horse which received an Eprex[registered sign] treatment during six consecutive days and a second one with a single injection of Aranesp[registered sign]. This inventive technology allowed the possibility to confirm the presence of rHuEPO within one day with a limit of detection validated for both urine and plasma at 250 pg mL-1 by means of a disposable, ready to use immunoaffinity column. The lower limit of detection (LLOD) obtained for each matrix was 100 pg mL-1. These results provide an important improvement for rHuEPO doping control in horseracing especially the possibility to confirm these banned molecules in both matrices, urine and plasma, with a confidence of two specific target peptides.
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| 7. |
- Baldassarre, Maurizio, et al.
(author)
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Pushing the detection limit of infrared spectroscopy for structural analysis of dilute protein samples.
- 2014
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In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 139:21, s. 5393-5399
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Journal article (peer-reviewed)abstract
- Fourier-transform infrared spectroscopy is a powerful and versatile tool to investigate the structure and dynamics of proteins in solution. The intrinsically low extinction coefficient of the amide I mode, the main structure-related oscillator, together with the high infrared absorptivity of aqueous media, requires that proteins are studied at high concentrations (>10 mg L(-1)). This may represent a challenge in the study of aggregation-prone proteins and peptides, and questions the significance of structural data obtained for proteins physiologically existing at much lower concentrations. Here we describe the development of a simple experimental approach that increases the detection limit of protein structure analysis by infrared spectroscopy. Our approach relies on custom-made filters to isolate the amide I region (1700-1600 cm(-1)) from irrelevant spectral regions. The sensitivity of the instrument is then increased by background attenuation, an approach consisting in the use of a neutral density filter, such as a non-scattering metal grid, to attentuate the intensity of the background spectrum. When the filters and grid are combined, a 2.4-fold improvement in the noise level can be obtained. We have successfully tested this approach using a highly diluted solution of pyruvate kinase in deuterated medium (0.2% w/v), and found that it provides spectra of a quality comparable to those recorded with a 10-fold higher protein concentration.
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| 8. |
- Baldassarre, Maurizio, et al.
(author)
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Simultaneous acquisition of infrared, fluorescence and light scattering spectra of proteins : direct evidence for pre-fibrillar species in amyloid fibril formation
- 2016
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In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 141:3, s. 963-973
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Journal article (peer-reviewed)abstract
- Different spectroscopic approaches are often used to probe specific aspects of amyloid fibril formation but are usually performed separately and under different conditions. This makes it problematic to relate different aspects of the aggregation process when these are monitored by different methods. We report on a multispectral approach for simultaneous acquisition of infrared, fluorescence and light scattering spectra of proteins undergoing aggregation. We have applied our approach to study beta-lactoglobulin, a milk protein known to form amyloid fibrils under well-established conditions. Our real-time multispectral measurements show that unfolding of this protein is followed by formation of early aggregates consisting of intermolecular beta-sheets with a typical infrared absorption at similar to 1619 cm(-1) in (H2O)-H-2. These aggregates, which lead to an increase in the light scattering signal, do not bind the amyloid-specific fluorophore ThT and therefore consist of oligomers or protofibrils. Fibril growth is then observed as a sigmoidal increase in ThT fluorescence. After similar to 25 h, a plateau is observed in the intensities of ThT emission and of the band at 1619 cm(-1), indicating that no new fibrils are forming. However, a second phase in the light scattering signal taking place after similar to 25 h suggests that the fibrils are assembling into larger structures, known as mature fibrils. This is associated with an upshift of the main beta-sheet band in the infrared spectrum. TEM analyses confirmed the existence of thick fibrils comprising 3-5 filaments.
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| 9. |
- Baldassarre, Maurizio, et al.
(author)
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The carbonate/bicarbonate system as a pH indicator for infrared spectroscopy
- 2014
-
In: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 139:9, s. 2167-2176
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Journal article (peer-reviewed)abstract
- Caged compounds capable of inducing large pH-jumps upon UV illumination have represented a breakthrough in time-resolved infrared spectroscopy of acidification-triggered phenomena, but their use is hampered by the inability to control the initial pH as well as to measure the final pH in mu L volumes. We have developed an experimental approach that accurately measures the initial and final pH values in pH-jump experiments. Our approach exploits the concomitant presence of two or more inorganic ions, such as carbonate and bicarbonate, that are added to the sample at a known concentration. The difference spectrum obtained in the infrared measurement is fitted to isolate the bands arising from the appearance or disappearance of either protonation state, and is then compared to a synthetic library of difference spectra generated using both qualitative (band position and width, extinction coefficient, pK) and quantitative (concentration, pathlength) parameters of the reporter ions. We have tested this approach in UV-photolysis experiments of 1-(2-nitrophenyl)ethyl sulfate in the presence of different concentrations of Na2CO3 and successfully used the infrared absorption of the carbonate and the bicarbonate ions to determine the initial and final pH values before and after the pH-jump, respectively.
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| 10. |
- Bassan, Paul, et al.
(author)
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The inherent problem of transflection-mode infrared spectroscopic microscopy and the ramifications for biomedical single point and imaging applications.
- 2013
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In: The Analyst. - : Royal Society of Chemistry (RSC). - 1364-5528 .- 0003-2654. ; 138:1, s. 144-57
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Journal article (peer-reviewed)abstract
- Transflection-mode FTIR spectroscopy has become a popular method of measuring spectra from biomedical and other samples due to the relative low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. In this publication we state an optical description of samples on multilayer low-e reflective substrates. Using this model we are able to explain in detail the so-called electric-field standing wave effect and rationalise the non-linear change in absorbance with sample thickness. The ramifications of this non-linear change, for imaging and classification systems, where a model is built from tissue sectioned at a particular thickness and compared with tissue of a different thickness are discussed. We show that spectra can be distorted such that classification fails leading to inaccurate tissue segmentation which may have subsequent implications for disease diagnostics applications.
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