Methods Protoc., Volume 1, Issue 1 (March 2018) – 9 articles

A new method for the measurement of accurate masses using direct infusion in an electrospray-triple quadrupole mass spectrometer is presented and compared to the traditional method using high-resolution mass spectrometry. The proposed method uses internal calibrants and post-acquisition calibration of the mass spectrum signal using the MassWorks software to determine accurate masses. Then, based on parameters such as elemental composition, number of double bond equivalents, and type of ion (even- or odd-electron), etc., a list of potential molecular formula candidates are generated and ranked according to spectral accuracy, (i.e., similarity between the calibrated profile and theoretical isotopic patterns). Experiments using six diverse synthesis products showed that mass accuracy in the Quattro Premier triple quadrupole mass spectrometer (QqQMS) was ≤9.2 mDa and spectral accuracy was ≥90.6%. According to both mass accuracy tolerance (±10 mDa) and spectral accuracy, the correct molecular formula was ranked in the top seven compounds out of up to 32 potential candidates. When considering the context of the synthesis reaction, only one formula was possible. In summary, results showed that the measurement of spectral accuracy in a low-resolution instrument such as the triple quadrupole was strongly dependent on the signal intensity and the presence of interfering peaks in the profile mass range window. This study suggests that use of triple quadrupole mass spectrometry followed by post-acquisition calibration can be an economical and robust approach compared to the traditional method using high-resolution mass spectrometers for the measurement of accurate masses in routine applications using small organic molecules at microgram-per-litter concentrations in relatively clean matrices. Full article

Pseudotype neutralization assays are powerful tools to study functional antibody responses against viruses in low biosafety laboratories. However, protocols described in the literature differ widely with respect to material, reagents, and methods used to perform these assays and to analyse the raw data generated. This could result in discrepancies between the results of different laboratories even when the same pseudotypes and the same samples are analysed. Here, we describe, in detail, an experimental protocol to perform pseudotype neutralization assays using lentiviral pseudotypes bearing influenza haemagglutinin and expressing firefly luciferase. We also present the steps necessary to analyse the data and calculate the half maximal inhibitory concentration of the sera analysed. This protocol will provide support for the validation and the standardization of the pseudotype neutralization assay for influenza virus serology. Additionally, it will provide a starting point for the development of pseudotype neutralization assays using pseudotypes bearing other viral envelope proteins. Full article

Positron Emission Tomography scan images are extensively used in radiotherapy planning, clinical diagnosis, assessment of growth and treatment of a tumor. These all rely on fidelity and speed of detection and delineation algorithm. Despite intensive research, segmentation has remained a challenging problem due to the diverse image content, resolution, shape, and noise. This paper presents a fast positron emission tomography tumor segmentation method using superpixels. Principal component analysis is applied on the superpixels and their average value. The distance vector of each superpixel from the average is computed in the principal components coordinate system. Finally, k-means clustering is applied on the distance vector to recognize tumor and non-tumor superpixels. The proposed approach is implemented in MATLAB 2016A, and promising accuracy with execution time of 2.35 ± 0.26 s is achieved. Fast execution time is achieved since the number of superpixels, and the size of distance vector on which clustering was done are low compared to the number of pixels in the image. Full article

The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth genomic characterization. It is essential to ascribe specific functions to phage genes, which will give information to unravel phage biology and to ensure the lack of undesirable genes, such as virulence and antibiotic resistance genes. Here, we describe a simple protocol for the selection of phage mutants carrying random deletions along the phage genome. Theoretically, any DNA region might be removed with the only requirement that the phage particle viability remains unaffected. This technique is based on the instability of phage particles in the presence of chelating compounds. A fraction of the phage population naturally lacking DNA segments will survive the treatment. Within the context of phages as antimicrobials, this protocol is useful to select lytic variants from temperate phages. In terms of phage efficiency, virulent phages are preferred over temperate ones to remove undesirable bacteria. This protocol has been used to obtain gene mutations that are involved in the lysogenic cycle of phages infecting Gram-positive bacteria (Staphylococcus and Lactobacillus). Full article

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) assisted generation of mutant animals has become the method of choice for the elucidation of gene function in development and disease due to the shortened timelines for generation of a desired mutant, the ease of producing materials in comparison to other methodologies (such as embryonic stem cells, ESCs) and the ability to simultaneously target multiple genes in one injection session. Here we describe a step by step protocol, from preparation of materials through to injection and validation of a cytoplasmic injection, which can be used to generate CRISPR mutants. This can be accomplished from start of injection to completion within 2–4 h with high survival and developmental rates of injected zygotes and offers significant advantages over pronuclear and other previously described methodologies for microinjection. Full article

A single solution protocol has been widely used for the fluorimetric determination of H₂O₂ in natural waters by its bleaching of the fluorescing scopoletin in the presence of the enzyme horseradish peroxidase (HRP). In this protocol, the reaction between scopoletin and H₂O₂ in the sample and the subsequent internal additions, and the measurements of the fluorescence are all carried out at a single pH in a fluorometer cell. It is found that this protocol is prone to four sources of possible error. The variability in the reaction stoichiometry between scopoletin and H₂O₂ in the presence of varying amounts of excess scopoletin, the effect of pH on the rate of reaction between scopoletin and H₂O₂, the photobleaching of scopoletin, and the de-activation of HRP. These possible sources of error can be circumvented in a two-stage protocol in which the reaction between H₂O₂ and scopoletin is carried out immediately upon sampling at a pH of 7, and the measurement of the fluorescence is carried out later on at a pH of 9. It should be the protocol of choice. Furthermore, in the two-stage protocol, after the initial reaction between H₂O₂ and scopoletin, the sample may be stored at room temperature for six days and at 4 °C for at least a month before its fluorescence is measured. This option can significantly reduce the logistics in the field. Full article

While many methods exist to quantitatively determine protein kinase activities, 32P-based radioactive assays remain the workhorse of many laboratories due to their high sensitivity, high signal to noise ratio, lack of interference by fluorescent and light-absorbing small molecules, and easy quantitation. Here, we demonstrate that the interaction between the yeast Rad53 Forkhead-associated (FHA) domain and a peptide optimized for phosphorylation by AMP-Activated Protein Kinase (AMPK), which has previously been exploited for the generation of intracellular phosphorylation sensors, can serve as a readout for a highly sensitive two-step AMPK AlphaScreen kinase assay with exceptional signal-to-noise ratio. Full article

The small heterobifunctional linker succinimidyl iodoacetate (SIA) was examined for the preparation of peptide–protein bioconjugates with predicable conjugation ratios. For many conjugation protocols, the protein is either treated with a reductant to cleave disulfide bonds or is reacted with thiolation chemicals, such as Traut’s reagent. Both approaches are difficult to control, need individual optimization and often lead to unsatisfactory results. In another popular approach, a heterobifunctional linker with a N-hydroxysuccinimide (NHS) and a maleimide functionality is applied to the protein. After the activation of some lysine ε-amino groups with the NHS ester functionality, a cysteine-containing peptide is attached to the activated carrier protein via maleimide. Particularly, the maleimide reaction leads to some unwanted byproducts or even cleavage of the linker. Many protocols end up with conjugates with unpredictable and irreproducible conjugation ratios. In addition, the maleimide-thiol addition product should be assumed immunogenic in vivo. To avoid these and other disadvantages of the maleimide approach, we examined the known linker succinimidyl iodoacetate (SIA) in more detail and developed two protocols, which lead to peptide–protein conjugates with predefined average conjugation ratios. This holds potential to eliminate tedious and expensive optimization steps for the synthesis of a bioconjugate of optimal composition. Full article