Mass spectrometry-based Cellular Thermal Shift Assay (CETSA®) for target deconvolution in phenotypic drug discovery

The recent renewed interest in phenotypic drug discovery has concomitantly put a focus on target deconvolution in order to achieve drug-target identification. Even though there are prescribed therapies whose mode of action is not fully understood, knowledge of the primary target will inevitably facilitate the discovery and translation of efficacy from bench to bedside. Elucidating targets and subsequent pathways engaged will also facilitate safety studies and overall development of novel drug candidates. Today, there are several techniques available for identifying the primary target, many of which rely on mass spectrometry (MS) to identify compound – target protein interactions. The Cellular Thermal Shift Assay (CETSA®) is well suited for identifying target engagement between ligands and their protein targets. Several studies have shown that CETSA combined with MS is a powerful technique that allows unlabeled target deconvolution in complex samples such as intact cells and tissues in addition to cell lysates and other protein suspensions. The applicability of CETSA MS for target deconvolution purposes will be discussed and exemplified in this mini review.     

Molecular genetic analysis of the polymorphic apolipoprotein E in modern and ancient human DNA samples

In this report, methodical bases for the molecular genetic analysis of the three common apolipoprotein E alleles APOE*2, APOE*3 and APOE*4 in DNA isolated from ancient human skeletal remains are described. Considering that ancient DNA target regions for amplification are generally quite small, the detection method is based on short amplification products in the range from 71 bp to 75 bp. The applicability of the modified method for APOE genotyping was examined in modern human DNA samples.     

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography,Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d(2)), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.     

A universal TaqMan-based RT-PCR protocol for cost-efficient detection of small noncoding RNA

Several methods for the detection of RNA have been developed over time. For small RNA detection, a stem–loop reverse primer-based protocol relying on TaqMan RT-PCR has been described. This protocol requires an individual specific TaqMan probe for each target RNA and, hence, is highly cost-intensive for experiments with small sample sizes or large numbers of different samples. We describe a universal TaqMan-based probe protocol which can be used to detect any target sequence and demonstrate its applicability for the detection of endogenous as well as artificial eukaryotic and bacterial small RNAs. While the specific and the universal probe-based protocol showed the same sensitivity, the absolute sensitivity of detection was found to be more than 100-fold lower for both than previously reported. In subsequent experiments, we found previously unknown limitations intrinsic to the method affecting its feasibility in determination of mature template RISC incorporation as well as in multiplexing. Both protocols were equally specific in discriminating between correct and incorrect small RNA targets or between mature miRNA and its unprocessed RNA precursor, indicating the stem–loop RT-primer, but not the TaqMan probe, triggers target specificity. The presented universal TaqMan-based RT-PCR protocol represents a cost-efficient method for the detection of small RNAs.     

The application of preirradiation combustion and neutron activation analysis technique for the determination of iodine in food and environmental reference materials

The pre-irradiation combustion (PC) of samples to liberate iodine, followed by trapping the iodine on charcoal and quantifying the element by neutron activation analysis (NAA), has been used at the National Institute of Standards and Technology for the determination of iodine in biological materials. The applicability of this technique to numerous environmental and dietary matrices is illustrated by analysis of a range of certified reference materials (CRMs) and a powdered grass material that was prepared as an in-house reference material (RM). Because of the combustion step involved, samples with low or no fat content (e.g., cereal products, selected botanical specimens, and nonfat milk powder) and inorganic materials (e.g., coal fly ash and dried sediments) are more suited for analysis by this method. In general, the results for several types of samples obtained by this method agreed with those obtained by a second radiochemical (R) NAA, as well as by a third method using inductively coupled plasma mass spectrometry (ICP-MS). PC-NAA is a useful technique for determining iodine in biological and environmental samples, especially for verification of iodine results obtained from other methods.     

General methods for the analysis of metabolic profiles of bile acids and related compounds in feces

A general method is described for the detailed qualitative and quantitative analysis of bile acids and related compounds from feces. The technique utilizes a novel combination of liquid-gel and liquid-solid extraction, lipophilic ion exchange chromatography, and capillary column gas-liquid chromatography coupled to mass spectrometry, which permits the detailed composition of bile acids in feces in terms of both the individual bile acids present and their mode of conjugation in the original fecal sample. The extraction, purification, and isolation procedures have been evaluated using fecal samples containing endogenous radioactive bile acid metabolites and from the addition of radiolabeled standards to fecal homogenates. The applicability of the general procedure is illustrated with examples from the analysis of bile acids and sterols in the feces collected from normal healthy subjects, patients with chronic diarrhea, and an adult female Sprague-Dawley rat. The flexibility of the method, and the general problems encountered in the extraction, purification, and isolation of bile acids and related classes of compounds from feces for subsequent analysis of gas-liquid chromatography are discussed in detail.     

Paper-based archiving of mammalian and plant samples for RNA analysis

The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.     

Use of polyvalent phage for reduction of streptomycetes on soil dilution plates.

An isolation method was developed in which prior to inoculation soil suspensions were exposed to suspensions of polyvalent phage isolated to Streptomyces spp. The phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates subsequent to inoculation, and this reduction was persistent after long incubation periods. The efficiency and applicability of the method developed were checked with different samples from a range of sources. The increased chances of development of other genera after the reduction of streptomycetes on soil dilution plates were assessed.     

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.     

Lysis and fractionation of Mycobacterium paratuberculosis and Escherichia coli by matrix solid-phase dispersion.

A novel method for the lysis and subsequent fractionation of bacterial constituents from Mycobacterium paratuberculosis strain 19698 (M. paratuberculosis) and Escherichia coli strain DH5 alpha utilizing the technique of matrix solid-phase dispersion (MSPD) is described. Bacteria were blended with octadecylsilyl (C18) derivatized silica to obtain cellular lysis. The blended material was used to prepare a column which was sequentially eluted with solvents of increasing polarity. Fractionation of cellular components was confirmed by analysis of the solvent extracts. The possible applicability of the MSPD technique as a general method for the lysis and fractionation of bacterial components is proposed.     

Optical Magnetism in Surface Plasmon Resonance–Based Sensors for Enhanced Performance

Surface plasmon resonance (SPR)–based structures are finding important applications in sensing biological as well as inorganic samples. In SPR techniques, an angle-resolved reflection (R) profile of the incident light from a metal-dielectric interface is measured and the resonance characteristics are extracted for the identification of the target sample. However, the performance, and hence the applicability of these structures, suffers when the weight and concentration of the target samples are small. Here, we show that SPR-based sensors can create strong magnetism at optical frequency, which can be used for the detection of target samples instead of using the conventional R profiles, as the magnetic resonance varies depending on the refractive index of the target sample. Using scattering parameters retrieval method, we computationally find out the effective permeability (μ_eff) of a SPR sensor with a structure based on Kretschmann configuration, and use it to calculate the performance of the sensor. A comparison with the conventional technique that uses R profile to detect a target sample shows a significant increase in the sensor performance when μ_eff is used instead.

     

Simple dual-spotting procedure enhances nLC-MALDI MS/MS analysis of digests with less specific enzymes

The beneficial effect of high mass accuracy in mass spectrometry is especially pronounced when using less specific enzymes as the number of theoretically possible peptides increases dramatically without any cleavage specificity defined. Together with a preceding chromatographic separation, high-resolution mass spectrometers such as the MALDI-LTQ-Orbitrap are therefore well suited for the analysis of protein digests with less specific enzymes. A combination with fast, automated, and informative MALDI-TOF/TOF analysis has already been shown to yield increased total peptide and protein identifications. Here, a simple method for nLC separation and subsequent alternating spotting on two targets for both a MALDI-LTQ-Orbitrap and a MALDI-TOF/TOF instrument is introduced. This allows for simultaneous measurements on both instruments and subsequent combination of both data sets by an in-house written software tool. The performance of this procedure was evaluated using a mixture of four standard proteins digested with elastase. Three replicate runs were examined concerning repeatability and the total information received from both instruments. A cytosolic extract of C. glutamicum was used to demonstrate the applicability to more complex samples. Database search results showed that an additional 32.3% of identified peptides were found using combined data sets in comparison to MALDI-TOF/TOF data sets.     

Automated protein identification by the combination of MALDI MS and MS/MS spectra from different instruments

The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.     

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson-Crick helices displaying different kinetic properties. The target-analyte B-C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target-analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target-analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.     

Specific on-plate enrichment of phosphorylated peptides for direct MALDI-TOF MS analysis

An on-plate specific enrichment method is presented for the direct analysis of peptides phosphorylation. An array of sintered TiO 2 nanoparticle spots was prepared on a stainless steel plate to provide porous substrate with a very large specific surface and durable functions. These spots were used to selectively capture phosphorylated peptides from peptide mixtures, and the immobilized phosphopeptides could then be analyzed directly by MALDI MS after washing away the nonphosphorylated peptides. beta-Casein and protein mixtures were employed as model samples to investigate the selection efficiency. In this strategy, the steps of phosphopeptide capture, purification, and subsequent mass spectrometry analysis are all successfully accomplished on a single target plate, which greatly reduces sample loss and simplifies analytical procedures. The low detection limit, small sample size, and rapid selective entrapment show that this on-plate strategy is promising for online enrichment of phosphopeptides, which is essential for the analysis of minute amount of samples in high-throughput proteome research.     

Deep learning image analysis models pretrained on daily objects are useful for the preliminary characterization of particulate pharmaceutical samples

Visible and subvisible particles are a quality attribute in sterile pharmaceutical samples. A common method for characterizing and quantifying pharmaceutical samples containing particulates is imaging many individual particles using high-throughput instrumentation and analyzing the populations data. The analysis includes conventional metrics such as the particle size distribution but can be more sophisticated by interpreting other visual/morphological features. To avoid the hurdles of building new image analysis models capable of extracting such relevant features from scratch, we propose using well-established pretrained deep learning image analysis models such as EfficientNet. We demonstrate that such models are useful as a prescreening tool for high-level characterization of biopharmaceutical particle image data. We show that although these models are originally trained for completely different tasks (such as the classification of daily objects in the ImageNet database), the visual feature vectors extracted by such models can be useful for studying different types of subvisible particles. This applicability is illustrated through multiple case studies: (i) particle risk assessment in prefilled syringe formulations containing different particle types such as silicone oil, (ii) method comparability with the example of accelerated forced degradation, and (iii) excipient influence on particle morphology with the example of Polysorbate 80 (PS80). As examples of agnostic applicability of pretrained models, we also elucidate the application to two high-throughput microscopy methods: microflow and background membrane imaging. We show that different particle populations with different morphological and visual features can be identified in different samples by leveraging out-of-the-box pretrained models to analyze images from each sample.