Inter‐laboratory comparison: Quantitative surface analysis of thin Fe‐Ni alloy films

An international interlaboratory comparison of the measurement capabilities of four National Metrology Institutes (NMIs) and one Designated Institute (DI) in the determination of the chemical composition of thin Fe‐Ni alloy films was conducted via a key comparison (K‐67) of the Surface Analysis Working Group of the Consultative Committee for Amount of Substance. This comparison was made using XPS (four laboratories) and AES (one laboratory) measurements. The uncertainty budget of the measured chemical composition of a thin alloy film was dominated by the uncertainty of the certified composition of a reference specimen which had been determined by inductively coupled plasma mass spectrometry using the isotope dilution method. Pilot study P‐98 showed that the quantification using relative sensitivity factors (RSFs) of Fe and Ni derived from an alloy reference sample results in much more accurate result in comparison to an approach using RSFs derived from pure Fe and Ni films. The individual expanded uncertainties of the participants in the K‐67 comparison were found to be between 2.88 and 3.40 atomic %. The uncertainty of the key comparison reference value (KCRV) calculated from individual standard deviations and a coverage factor (k) of 2 was 1.23 atomic %. Copyright © 2011 John Wiley & Sons, Ltd.     

Time-based analysis of silver-stained proteins in acrylamide gels

Silver staining of proteins after PAGE often remains the method of choice in many laboratories. Nevertheless, it is known that quantification of protein levels is keenly restricted to a small range of protein concentrations leading to an over- or underestimation of protein amounts. To overcome this, a time-based analysis method was developed to avoid the saturation effect of the silver-staining reaction, thus resulting in an improved dynamic range of the gel image produced and therefore better quantification of proteins. Instead of the well-known end-point image analysis, gray intensities of time series images of a developing gel are determined and times until a threshold gray value is reached are calculated. These times are used to calculate a new grayscale image which can be analyzed using commercial image processing software.     

A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors

Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 microm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties.     

Validated multi-component CZE-UV procedure for the quantification of human hemorphin LVV-H7 in plasma stability studies

The human hemorphin LVV-H7 is an endogenous cleavage product of the hemoglobin β, γ, ε or δ chain exhibiting potential pharmaceutical relevance for blood pressure regulation, the treatment of Alzheimer’s disease or learning deficiencies. Here we present the development of a multi-component capillary zone electrophoretic method (CZE-UV), allowing the simultaneous quantification of LVV-H7 and four N-terminal degradation products generated in EDTA plasma. Hemorphins in the supernatant of precipitated plasma samples are quantified by external calibration. Validation of the procedure oriented towards international pharmaceutical guidelines and demonstrated excellent linearity ( r ² ≥0.999), good precision (repeatability and reproducibility below 11%), accuracy (−8.4%–4%), ruggedness and an appropriate lower limit of quantification (LLOQ 1.0 μg mL⁻¹). This procedure was applied to stability studies of LVV-H7 in human EDTA plasma attended by profiling metabolites using qualitative MALDI-TOF MS analysis. We detected the activity of a soluble plasma form of aminopeptidase M causing successive N-terminal truncation. This is the first time that LVV-H7 degradation as well as its metabolite production have systematically been monitored by a quantitative CZE-UV procedure, underlining the growing importance of such techniques in peptide analysis. In addition, our results give useful hints for future drug development of LVV-H7.     

Isotope coded protein label quantification of serum proteins—Comparison with the label-free LC-MS and validation using the MRM approach

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids - serum - and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM based protein quantification technique. Regarding the ICPL approach, serum samples used in this study were depleted of high abundant proteins, labeled with ICPL and fractionated according to their respective pI (3-5, 5-7 and 7-12). The samples were further subjected to tryptic digestion followed by treatment with the Glu-C enzyme. The peptides were analyzed on a 2D-nano-LC system using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The LC system was connected on-line with the electrospray ion-trap mass spectrometer. For the label-free quantification the serum samples were depleted and digested with trypsin. A proteome-wide comparison was performed using highly reproducible LC and data independent alternate scanning in conjunction with a high mass accuracy orthogonal time-of-flight mass spectrometer. Selected proteins, found by both methods, were validated using the MRM approach. For this purpose non-depleted tryptically digested serum samples were analyzed by LC coupled with a triple-quadrupole MS. The relative protein quantification using ICPL and mass spectrometry allowed for the detection of approximately 200 proteins, whereas about 2/3 of those contained the ICPL label and could therefore be quantified. Label-free approach used no fractionation, less sample and was able to identify and quantify over 110 proteins. The identified proteins covered generally 3-4 orders of magnitude of protein concentration in human serum. Changes in relative abundance of eight proteins were validated using MRM. This study, for the first time, shows the ability of the relative protein quantification based upon ICPL and 2D-LC-MS/MS to quantify serum biomarkers. It provides two additional label-free approaches that could validate and bring additional value to the label-based results, offering a starting point for comprehensive proteomics studies aiming at revealing biomarkers of clinical relevance.     

Decision peptide-driven: A free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry

The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse ¹⁸O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse ¹⁸O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse ¹⁸O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using ¹⁸O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase.     

A Stable Isotopic Pre-digestion Labeling Method for Protein Quantitative Analysis Using Matrix-assisted Laser Desorption/ionization Mass Spectrometry

As an extension of our previous work, here a strategy was demonstrated for protein identification and quantification analyses utilizing a combination of stable isotope chemical labeling with subsequent denaturation, enzymatic digestion and matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Using [d₀]‐ and [d₆]‐4,6‐dimethoxy‐2‐(methylsulfonyl)pyrimidine ([d₀]‐/[d₆]‐DMMSP), stable isotopic labels were incorporated before digestion. The comparative samples were combined before labeling after digestion, thus biases resulting from differences in sample digestion were avoided and the higher accuracy of quantification could be attained. The labeling was spatial‐selective to particular residues of cysteine, lysine, and tyrosine before denaturation, which could lead to a better universality of the strategy for cysteine‐free proteins. In addition, some lysine residues were blocked after labeling, the partly destroyed recognition sites could simplify the trypsin hydrolysates and hence facilitate the MS complexity. Together, our one‐step labeling strategy combined several desirable properties such as spatial‐selective labeling, reliability of quantitative results, simplification of analysis of complex systems and direct analysis with minimum sample handling. Our results demonstrate the usefulness of the method for analyzing lysozyme in egg white. The method was expected to provide a new powerful tool for comparative proteome research.     

HPLC-CHIP coupled to a triple quadrupole mass spectrometer for carbonic anhydrase II quantification in human serum

A method for carbonic anhydrase II (CA II) absolute quantification in human serum is presented. This method is based on high-performance liquid chromatography (HPLC)-Chip microfluidic device incorporating a nanoelectrospray source interfaced to a triple quadrupole mass spectrometer. The fraction containing CA II was isolated by preparative reversed-phase HPLC, and peptides obtained from the tryptic digest of the protein mixture were separated by the HPLC-Chip system. The multiple-reaction monitoring acquisition mode of a selected suitable CA II peptide and peptide internal standard allowed the selective and sensitive determination of a CA II. Absolute recovery of the method was 52 ± 12%, while analytical recovery was 81 ± 10%. For the eight samples analyzed, the matrix effect was found to be only −14 ± 6%. A comparison among three regression lines type which were obtained by external calibration, matrix-matched calibration, and standard addition method, respectively, demonstrated that the first one is adequate in obtaining good accuracy and precision. Method quantification limit for CA II in serum was estimated to be 2 fmol/mL. CA II mean concentration in sera from eight healthy subjects was found to be 56 pmol/mL (relative standard deviation 24%).     

Dynamics of Remote Communication: Movement Coordination in Video-Mediated and Face-to-Face Conversations

The present pandemic forced our daily interactions to move into the virtual world. People had to adapt to new communication media that afford different ways of interaction. Remote communication decreases the availability and salience of some cues but also may enable and highlight others. Importantly, basic movement dynamics, which are crucial for any interaction as they are responsible for the informational and affective coupling, are affected. It is therefore essential to discover exactly how these dynamics change. In this exploratory study of six interacting dyads we use traditional variability measures and cross recurrence quantification analysis to compare the movement coordination dynamics in quasi-natural dialogues in four situations: (1) remote video-mediated conversations with a self-view mirror image present, (2) remote video-mediated conversations without a self-view, (3) face-to-face conversations with a self-view, and (4) face-to-face conversations without a self-view. We discovered that in remote interactions movements pertaining to communicative gestures were exaggerated, while the stability of interpersonal coordination was greatly decreased. The presence of the self-view image made the gestures less exaggerated, but did not affect the coordination. The dynamical analyses are helpful in understanding the interaction processes and may be useful in explaining phenomena connected with video-mediated communication, such as “Zoom fatigue”.     

土壤样品中微塑料的分析方法研究进展

微塑料作为一种新型污染物,因其粒径小、难以降解、本身含有多种污染物、且易吸附疏水性有机污染物和重金属等产生复合污染,已成为国内外学者研究的热点问题.微塑料广泛存在于土壤环境中,进入土壤中的微塑料会影响土壤理化性质,同时影响土壤动物生长、植物生长和微生物活动等,并且,微塑料会在土壤中发生迁移或沿食物链传递从而对人类健康构...