In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells.

Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.

In-Needle Pre-Column Derivatization for Amino Acid Quantification (iPDAQ) Using HPLC.

Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor.

Comparative Evaluation of Plasma Metabolomic Data from Multiple Laboratories.

In mass spectrometry-based metabolomics, the differences in the analytical results from different laboratories/machines are an issue to be considered because various types of machines are used in each laboratory. Moreover, the analytical methods are unique to each laboratory. It is important to understand the reality of inter-laboratory differences in metabolomics. Therefore, we have evaluated whether the differences in analytical methods, with the exception sample pretreatment and including metabolite extraction, are involved in the inter-laboratory differences or not. In this study, nine facilities are evaluated for inter-laboratory comparisons of metabolomic analysis. Identical dried samples prepared from human and mouse plasma are distributed to each laboratory, and the metabolites are measured without the pretreatment that is unique to each laboratory. In these measurements, hydrophilic and hydrophobic metabolites are analyzed using 11 and 7 analytical methods, respectively. The metabolomic data acquired at each laboratory are integrated, and the differences in the metabolomic data from the laboratories are evaluated. No substantial difference in the relative quantitative data (human/mouse) for a little less than 50% of the detected metabolites is observed, and the hydrophilic metabolites have fewer differences between the laboratories compared with hydrophobic metabolites. From evaluating selected quantitatively guaranteed metabolites, the proportion of metabolites without the inter-laboratory differences is observed to be slightly high. It is difficult to resolve the inter-laboratory differences in metabolomics because all laboratories cannot prepare the same analytical environments. However, the results from this study indicate that the inter-laboratory differences in metabolomic data are due to measurement and data analysis rather than sample preparation, which will facilitate the understanding of the problems in metabolomics studies involving multiple laboratories.

Performance of functionalized monolithic silica capillary columns with different mesopore sizes using radical polymerization of octadecyl methacrylate.

We report on the possibility to enhance the phase ratio and retention factor in silica monoliths. According to pioneering work done by Núñez et al. [1], this enhancement is pursued by applying a stationary phase layer via radical polymerization with octadecyl methacrylate (ODM) as an alternative to the customary octadecylsilylation (C18-derivatization). The difference in band broadening, retention factor and separation selectivity between both approaches was compared. Different hydrothermal treatment temperatures for the column preparation were applied to produce monolithic silica structures with three different mesopore sizes (resp. 10, 13, and 16 nm, as determined by argon physisorption) while maintaining similar domain size (sum of through-pore and skeleton size). It has been found that the columns with the poly(octadecyl methacrylate)-phase (ODM columns) provided a 60 to 80% higher retention factor in methanol-water mixture compared to the octadecylsilylated (ODS) columns produced by starting from similar silica backbone structures. In acetonitrile-water mixture, the enhancement is smaller (15 to 30% times higher), yet significant. By adjusting the fabrication conditions (for both the preparation of the monolithic backbones and the surface functionalization), the achieved retention factors (up k = 4.89 for pentylbenzene in 80:20% (v/v) methanol/water) are obviously higher than obtained in the pioneering study on ODM monoliths of Núñez et al. [1], and column clogging could be completely avoided. In addition, also separation efficiencies were significantly higher than shown in Ref. [1], with plate heights as low as 5.8 µm. These plate heights are however inferior to those observed on the ODS-modified sister columns. The difference can be explained by the slower intra-skeleton diffusion displayed by the ODM-modified columns, in turn caused by the larger obstruction to diffusion originating from the thicker stationary phase layer.

Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing.

Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with ß-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.

Calibration-Curve-Locking Database for Semi-Quantitative Metabolomics by Gas Chromatography/Mass Spectrometry.

Calibration-Curve-Locking Databases (CCLDs) have been constructed for automatic compound search and semi-quantitative screening by gas chromatography/mass spectrometry (GC/MS) in several fields. CCLD felicitates the semi-quantification of target compounds without calibration curve preparation because it contains the retention time (RT), calibration curves, and electron ionization (EI) mass spectra, which are obtained under stable apparatus conditions. Despite its usefulness, there is no CCLD for metabolomics. Herein, we developed a novel CCLD and semi-quantification framework for GC/MS-based metabolomics. All analytes were subjected to GC/MS after derivatization under stable apparatus conditions using (1) target tuning, (2) RT locking technique, and (3) automatic derivatization and injection by a robotic platform. The RTs and EI mass spectra were obtained from an existing authorized database. A quantifier ion and one or two qualifier ions were selected for each target metabolite. The calibration curves were obtained as plots of the peak area ratio of the target compounds to an internal standard versus the target compound concentration. These data were registered in a database as a novel CCLD. We examined the applicability of CCLD for analyzing human plasma, resulting in time-saving and labor-saving semi-qualitative screening without the need for standard substances.

Nano-scale analyses of the chromatin decompaction induced by histone acetylation.

The acetylation of histone tails is a key factor in the maintenance of chromatin dynamics and cellular homeostasis. The hallmark of active chromatin is the hyper-acetylation of histones, which appears to result in a more open chromatin structure. Although short nucleosomal arrays have been studied, the structural dynamics of relatively long acetylated chromatin remain unclear. We have analyzed in detail the structure of long hyper-acetylated chromatin fibers using atomic force microscopy (AFM). Hyper-acetylated chromatin fibers isolated from nuclei that had been treated with Trichostatin A (TSA), an inhibitor of histone deacetylase, were found to be thinner than those from untreated nuclei. The acetylated chromatin fibers were more easily spread out of nuclei by high-salt treatment, implying that hyper-acetylation facilitates the release of chromatin fibers from compact heterochromatin regions. Chromatin fibers reconstituted in vitro from core histones and linker histone H1 became thinner upon acetylation. AFM imaging indicated that the gyration radius of the nucleosomal fiber increased after acetylation and that the hyper-acetylated nucleosomes did not aggregate at high salt concentrations, in contrast to the behavior of non-acetylated nucleosomal arrays, suggesting that acetylation increases long-range repulsions between nucleosomes. Based on these data, we considered a simple coarse grained model, which underlines the effect of remaining electric charges inside the chromatin fiber.

An Analytical System for Single-Cell Metabolomics of Typical Mammalian Cells Based on Highly Sensitive Nano-Liquid Chromatography Tandem Mass Spectrometry.

The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10-20 µm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-µm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 µm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3-132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabolites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity.

Dynamic Metabolome Analysis Reveals the Metabolic Fate of Medium-Chain Fatty Acids in AML12 Cells.

Several studies in hepatocyte cell lines reported that medium-chain fatty acids (MCFAs) with 6-12 carbons showed different metabolic properties from long-chain fatty acids (LCFAs). However, these studies reported unclear effects of different fatty acid molecules on hepatocyte metabolism. This study is aimed to capture the metabolic kinetics of MCFA assimilation in AML12 cells treated with octanoic acid (FA 8:0), decanoic acid (FA 10:0), or lauric acid (FA12:0) [LCFA; oleic acid (FA 18:1)] via metabolic profiling and dynamic metabolome analysis with 13C-labeling. The concentrations of total ketone bodies in the media of cells treated with FA 8:0 or FA 10:0 were 3.22- or 3.69-fold higher than those obtained with FA 18:1 treatment, respectively. FA 12:0 treatment did not significantly increase ketone body levels compared to DMSO treatment (control), whereas FA 12:0 treatment increased intracellular triacylglycerol (TG) levels 15.4 times compared to the control. Metabolic profiles of FA 12:0-treated samples differed from those of the FA 8:0-treated and FA 10:0-treated samples, suggesting that metabolic assimilation of MCFAs differed significantly depending on the MCFA type. Furthermore, the dynamic metabolome analysis clearly revealed that FA 8:0 was rapidly and quantitatively oxidized to acetyl-CoA and assimilated into ketone bodies, citrate cycle intermediates, and glucogenic amino acids but not readily into TGs.

Performance of small-domain monolithic silica columns in nano-liquid chromatography and comparison with commercial packed bed columns with 2 µm particles.

We report on a direct comparison of the separation performance in capillary nano-LC between commercial packed bed columns and the small-domain silica monoliths in applications. Octadecylsilylated monolithic silica capillary columns with a 50 and 100 µm inner diameter (i.d.) were prepared with a procedure providing domain sizes in the sub-2 µm range. The fabricated monolith columns could provide plate heights (H) of 4.0‒4.2 µm for hexylbenzene (retention factor (k) = 3.6) at an optimal linear velocity range under an isocratic condition, while showing a column permeability (Kv0 = 1.6‒1.8 × 10-14 m2) comparable to that of a column packed with 3‒3.5 µm particles. When the peak capacity (np) for a cytochrome C digest was compared for variable gradient times (tG = 15, 30, 60, and 120 min) and constant gradient steepness (b'), the present monolith columns could show a 30‒40% higher np-value than the packed capillary column with 2 µm particles (e.g. np = 180 versus np = 259 at tG = 30 min). The produced monolith columns showed a high chromatographic repeatability for both isocratic and gradient elution (e.g. relative standard deviation (n = 3, RSD (%)) = 0.5% for H, 2,6% for k, and 5.6% for Kv0 in the isocratic mode using the 100 µm i.d.-columns). The present results show that the domain sizes which can now be achieved for capillary silica monoliths are sufficiently small to result in separation efficiencies that can successfully compete with the commercial packed bed columns available for use in nano-LC applications.

Inter-Laboratory Comparison of Metabolite Measurements for Metabolomics Data Integration.

BACKGROUND: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory. METHODS: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory. RESULTS: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference. CONCLUSION: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization.

Silica-based hybrid porous layers to enhance the retention and efficiency of open tubular capillary columns with a 5 µm inner diame9ter.

We report on possibility to enhance the hydrophobicity of octadecylsilylated silica-based porous layered open tubular (PLOT) columns with an inner diameter (i.d.) of 5 µm by applying hybrid tetramethoxysilane (TMOS)/methyltrimethoxysilane (MTMS) layers with inserted methyl groups. Due to this higher hydrophobicity, thinner porous layers suffice to achieve similar retention factor (k) as in octadecylsilylated silica-based PLOT columns synthesized using TMOS only. Since thinner layers have a lower intra-layer mass transfer resistance, this in turn allows to obtain superior column efficiencies in comparison with separations carried out with TMOS-based PLOT columns at the same retention. Since layer thickness contributes to the C-term type of band broadening, this is most pronounced at high velocities. Typical gains in column efficiency at a reduced velocity of νi = 30 are on the order of 15%. Preparing the hybrid PLOT columns in 5 µm i.d.-capillaries with a length of 0.4 m using different TMOS/MTMS preparation mixtures leads to different layer thickness in the capillaries. It is shown that column efficiencies for the most retained compound (k = 0.9-1.5) went from N = 101,000 for PLOT columns with a layer thickness (df) of 250 nm, over N = 95,000 for df = 320 nm to N = 89,000 for df = 400 nm, corresponding to plate heights (H) in the order of 3.5-3.9 µm (reduced plate heights (h = 0.8-1.0)). By applying the same preparation mixtures for much longer capillaries of 1.3 m, a high repeatability of the volumetric phase ratio (m) (difference <1%) and the k-values (difference <5%) was observed between the 0.4 m and 1.3 m PLOT columns. In addition, also a very similar band broadening was obtained, as the h-values in the longer columns coincided well (order of a few % difference) with the reduced plate height curves measured in the shorter columns. The effect of the retention factor and layer thickness on these reduced plate height curves furthermore fits well with the Golay-Aris theory. Depending on the layer thickness, plate numbers in the longer capillary columns were varying from N = 282,000 to N = 379,000 for the most retained compound.