50 research outputs found
Besugárzás hatására zsĂrsavakbĂłl keletkezĹ‘ 2-alkil-ciklobutanonok kimutatása
A besugárzottság kimutatására használatos módszerek közül a szerzők a 2-alkil-ciklobutanonok meghatározására szolgáló eljárást ismertetik. Értékelik az Európai Unió szabványa szerinti kimutatás módszerét és az ismertté vált irányokat. A továbblépés feladatai közül kiemelést érdemel a szuperkritikus extrakció optimálása és a kimutatási határ csökkentése. From the methods used for the detection of being irradiated, a procedure for detection of 2-alkyl cyclobutanones is reported. The detection method according to the European Union standard and the research directions published so far are evaluated. From the tasks of further development, optimisation of the supercritical extraction and the lowering of detection limit is stressed. Von den für den Nachweis der Bestrahlung verwendeten Methoden wird das Verfahren zum Nachweis von 2-Alkyl-Cyklobutanon beschrieben. Die Standardmethode der Europäischer Union und die bekannt gewordenen Forschungsrichtungen werden diskutiert. Von den Aufgaben der Weiterentwicklung sind die Optimierung der superkritischen Extraktion und die Verringerung der Nachweisgrenzen hervorzuheben
Cigarette smoking prior to blood sampling acutely affects serum levels of the chronic obstructive pulmonary disease biomarker surfactant protein D
Dynamic binning peak detection and assessment of various lipidomics liquid chromatography-mass spectrometry pre-processing platforms
Liquid chromatography-mass spectrometry (LC-MS)-based lipidomics generates large datasets that need to be interpreted using high-performance data pre-processing tools such as XCMS, mzMine, and Pro genesis. These pre-processing tools rely heavily on accurate peak detection, which depends on proper setting of the peak detection mass tolerance (PDMT). The PDMT is usually set with a fixed value in either ppm or Da units. However, this fixed value may result in duplicates or missed peak detection and inaccurate peak quantification. To improve the accuracy of peak detection, we developed the dynamic binning method, which considers peak broadening described by the physics of ion separation and sets (m)(2) the PDMT dynamically in function of m/z. In our method, the PDMT is proportional to z for Fourier (m)(15) transform ion cyclotron resonance (FTICR), to z for Orbitrap and to m/z for Quadrupole time-of flight (Q-TOF), and is a constant for Quadrupole mass analyzer. The dynamic binning method was implemented in XCMS [1,2], and the adopted source code is available in GitHub at https://github.com/xiaodfeng/DynamicXCMS. We have compared the performance of the XCMS implemented dynamic binning with different popular lipidomics pre-processing tools to find differential compounds. We generated set samples with 43 lipid internal standards that were differentially spiked to aliquots of one human plasma lipid sample using Orbitrap LC-MS/MS. The performance of various pipelines using matched parameter sets was quantified by a quality score system that reflects the ability of a preprocessing pipeline to detect differential peaks spiked at various concentrations. The quality score indicated that our dynamic binning method improves the quantification performance of XCMS (maximum p-value 9.8$10-3 of two-sample Wilcoxon test) over its original implementation. We also showed that the XCMS with dynamic binning found differential spiked-in lipids better or with similar performance as mzMine and Progenesis do. (C) 2021 The Author(s). Published by Elsevier B.V
Imaging of protein distribution in tissues using mass spectrometry: An interdisciplinary challenge
The recent development of mass spectrometry imaging (MSI) technology allowed to obtain extremely detailed images of the spatial distribution of proteins in tissue at high spatial resolution reaching cell dimensions, high target specificity and a large dynamic concentration range. This review focusses on the development of two main MSI principles, namely targeted and untargeted detection of protein distribution in tissue samples, with special emphasis on the improvements in analyzed mass range and spatial resolution over the last 10 years. Untargeted MSI of in situ digested proteins with matrix-assisted laser desorption ionization is the most widely used approach, but targeted protein MSI technologies using laser ablation inductively coupled plasma (LA-ICP) and photocleavable mass tag chemical labeling strategies are gaining momentum. Moreover, this review also provides an overview of the effect of sample preparation on image quality and the bioinformatic challenge to identify proteins and quantify their distribution in complex MSI data
Tutorial: Correction of shifts in single-stage LC-MS(/MS) data
Abstract Label-free LC-MS(/MS) provides accurate quantitative profiling of proteins and metabolites in complex biological samples such as cell lines, tissues and body fluids. A label-free experiment consists of several LC-MS(/MS) chromatograms that might be acquired over several days, across multiple laboratories using different instruments. Single-stage part (MS1 map) of the LC-MS(/MS) contains quantitative information on all compounds that can be detected by LC-MS(/MS) and is the data of choice used by quantitative LC-MS(/MS) data pre-processing workflows. Differences in experimental conditions and fluctuation of analytical parameters influence the overall quality of the MS1 maps and are factors hampering comparative statistical analyses and data interpretation. The quality of the obtained MS1 maps can be assessed based on changes in the two separation dimensions (retention time, mass-to-charge ratio) and the readout (ion intensity) of MS1 maps. In this tutorial we discuss two types of changes, monotonic and non-monotonic shifts, which may occur in the two separation dimensions and the readout of MS1 map. Monotonic shifts of MS1 maps can be corrected, while non-monotonic ones can only be assessed but not corrected, since correction would require precise modelling of the underlying physicochemical effects, which would require additional parameters and analysis. We discuss reasons for monotonic and non-monotonic shifts in the two separation dimensions and readout of MS1 maps, as well as algorithms that can be used to correct monotonic or to assess the extent non-monotonic shifts. Relation of non-monotonic shift with peak elution order inversion and orthogonality as defined in analytical chemistry is discussed. We aim this tutorial for data generator and evaluators scientists who aim to known the condition and approaches to produce and pre-processed comparable MS1 maps
Assessment of sample preparation bias in mass spectrometry-based proteomics
For mass spectrometry-based proteomics, the selected sample preparation strategy is a key determinant for information that will be obtained. However, the corresponding selection is often not based on a fit-for-purpose evaluation. Here we report a comparison of in-gel (IGD), in-solution (ISD), on-filter (OFD), and on-pellet digestion (OPD) workflows on the basis of targeted (QconCAT-multiple reaction monitoring (MRM) method for mitochondrial proteins) and discovery proteomics (data dependent acquisition, DDA) analyses using three different human head and neck tissues (i.e. nasal polyps, parotid gland, and palatine tonsils). Our study reveals differences between the sample preparation methods, for example with respect to protein and peptide losses, quantification variability, protocol-induced methionine oxidation and asparagine/glutamine deamidation as well as identification of cysteine containing peptides. However, none of the methods performed best for all types of tissues, which argues against the existence of a universal sample preparation method for proteome analysis
Affimers as an Alternative to Antibodies in an Affinity LC-MS Assay for Quantification of the Soluble Receptor of Advanced Glycation End-Products (sRAGE) in Human Serum
Antibodies are indispensable tools in biomedical research, but their size, complexity, and sometimes lack of reproducibility created a need for the development of alternative binders to overcome these limitations. Affimers are a novel class of affinity binders based on a structurally robust protease inhibitor scaffold (i.e. Cystatin A), which are selected by phage display and produced in a rapid and simple E. coli protein expression system. These binders have a defined amino acid sequence with defined binding regions and are versatile thereby allowing for easy engineering. Here we present an affimer-based liquid chromatography-mass spectrometry (LC-MS) method for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker for chronic obstructive pulmonary disease (COPD). The method was validated according to European Medicines Agency and U.S. Food and Drug Administration guidelines and enabled quantitation of serum sRAGE between 0.2 and 10 ng/mL. Comparison between the affimer-based method and a previously developed, validated antibody-based method showed good correlation (R2 = 0.88), and indicated that 25% lower sRAGE levels are reported by the affimer-based assay. In conclusion, we show the first-time application of affimers in a quantitative LC-MS method, which supports the potential of affimers as robust alternatives to antibodies
Quantification of surfactant protein D (SPD) in human serum by liquid chromatography-mass spectrometry (LC-MS)
Quantification of intact proteins in complex biological matrices by liquid chromatography-mass spectrometry (LC-MS) is a promising analytical strategy but is technically challenging, notably for concentrations at or below the ng/mL level. Therefore, MS-based protein quantification is mostly based on measuring protein-specific peptides, so-called 'surrogate peptides', that are released through proteolysis. While quantitative protein bioanalysis based on peptide LC-MS is much more sensitive, not every peptide is suitable in this respect. For example, some peptides are too small to be unique for a protein while others are too large to be measured with sufficient sensitivity, so careful selection of appropriate peptides is essential. Here we present a validated LC-MS method for quantification of surfactant protein D (SPD) at clinically relevant levels between 5 and 500 ng/mL using 50 mu L. of serum. This method targets two SPD-specific peptides in the C-type lectin, ligand binding domain of the SPD protein. One of these peptides contains a methionine residue which would typically be avoided because of its unstable nature. Some quantitative methods do target methionine-containing peptides, and corresponding workflows feature an oxidation step at the peptide level using hydrogen peroxide (H2O2) to convert all methionine residues to more stable methionine sulfoxides. For our method, such a procedure was associated with peptide loss, hence we developed an oxidation procedure at the protein level using H2O2 to oxidize methionine residues and the enzyme catalase to quench excess H2O2. This procedure may be applicable to other quantitative methods based on a surrogate peptide-based approach and may potentially also be useful for MS-based workflows targeting intact proteins
Quantification of the soluble Receptor of Advanced Glycation End-Products (sRAGE) by LC-MS after enrichment by strong cation exchange (SCX) solid-phase extraction (SPE) at the protein level
The study of low abundant proteins contributes to increasing our knowledge about (patho) physiological processes and may lead to the identification and clinical application of disease markers. However, studying these proteins is challenging as high-abundant proteins complicate their analysis. Antibodies are often used to enrich proteins from biological matrices prior to their analysis, though antibody-free approaches have been described for some proteins as well. Here we report an antibody-free workflow on the basis of strong cation exchange (SCX) enrichment and liquid chromatography-mass spectrometry (LC-MS) for quantification of the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). sRAGE was quantified in serum at clinically relevant low to sub ng mL(-1) levels. The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and was compared to an antibody-based LC-MS sRAGE method. The SCX-based method builds upon the bipolar charge distribution of sRAGE, which has a highly basic N-terminal part and an acidic C-terminal part resulting in an overall neutral isoelectric point (pI). The highly basic N-terminal part (pI(calculated) = 10.3) allowed for sRAGE to be enriched by SCX at pH 10, a pH at which most serum proteins do not bind. This study shows that ion exchange-based enrichment is a viable approach for the LC-MS analysis of several low abundant proteins following a thorough analysis of their physical-chemical properties. (C) 2018 The Authors. Published by Elsevier B.V
Adsorptive Microtiter Plates As Solid Supports in Affinity Purification Workflows
Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-ÎşB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research