Simultaneous discovery of compounds dominated by either molding kinetics or geographical region of origin for moisture damaged cacao beans using orthogonally applied tile-based fisher ratio analysis of GC×GC-TOFMS data.

Herein, two "orthogonal" characteristics of moisture damaged cacao beans (temporally dependent molding kinetics versus the time-independent geographical region of origin) are simultaneously analyzed in a comprehensive two-dimensional (2D) gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) dataset using tile-based Fisher ratio (F-ratio) analysis. Cacao beans from six geographical regions were analyzed once a day for six days following the initiation of moisture damage to trigger the molding process. Thus, there are two "extremes" to the experimental sample class design: six time points for the molding kinetics versus the six geographical regions of origin, resulting in a 6 × 6 element signal array referred to as a composite chemical fingerprint (CCF) for each analyte. Usually, this study would involve initial generation of two separate hit lists using F-ratio analysis, one hit list from inputting the data with the six time point classes, then another hit list from inputting the dataset from the perspective of geographic region of origin. However, analysis of two separate hit lists with the intent to distill them down to one hit list is extremely time-consuming and fraught with shortcomings due to the challenges associated with attempting to match analytes across two hit lists. To address this challenge, tile-based F-ratio analysis is "orthogonally applied" to each analyte CCF to simultaneously determine two F-ratios at the chromatographic 2D location (F-ratiokinetic and F-ratioregion) for each hit, by ranking a single hit list using the higher of the two F-ratios resulting in the discovery of 591 analytes. Further, using a pseudo-null distribution approach, at the 99.9% threshold over 400 analytes were deemed suitable for PCA classification. Using a more stringent 99.999% threshold, over 100 analytes were explored more deeply using PARAFAC to provide a purified mass spectrum.

Evaluation of Methylotrophic Yeast Ogataea thermomethanolica TBRC 656 as a Heterologous Host for Production of an Animal Vaccine Candidate.

Multiple yeast strains have been developed into versatile heterologous protein expression platforms. Earlier works showed that Ogataea thermomethanolica TBRC 656 (OT), a thermotolerant methylotrophic yeast, can efficiently produce several industrial enzymes. In this work, we demonstrated the potential of this platform for biopharmaceutical manufacturing. Using a swine vaccine candidate as a model, we showed that OT can be optimized to express and secrete the antigen based on porcine circovirus type 2d capsid protein at a respectable yield. Crucial steps for yield improvement include codon optimization and reduction of OT protease activities. The antigen produced in this system could be purified efficiently and induce robust antibody response in test animals. Improvements in this platform, especially more efficient secretion and reduced extracellular proteases, would extend its potential as a competitive platform for biopharmaceutical industries.

Computational method for untargeted determination of cycling yeast metabolites using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry.

A computational method for the untargeted determination of cycling yeast metabolites using a comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) dataset is presented. The yeast metabolomic cycle for the diploid yeast strain CEN.PK with a 5 h cycle period relative to the O2 concentration level is comprehensively examined to determine the metabolites that exhibit cycling. Samples were collected over only two cycles (10 h with a total of 24 time-point sampling intervals at 25 min each) as an experimental constraint. Due to the limited number of cycles expressed in the dataset, a computational method was devised to determine with statistical significance whether or not a given metabolite exhibited a temporal signal pattern that constituted cycling in the context of the 5 h cycle period. The computational method we report compares the experimentally obtained 24 time-point metabolite signal sequences to randomly generated signal sequences coupled with statistically based confidence level LOF metrics to determine whether or not a given metabolite expresses cycling, and if so, what is the phase of the cycling. Initially the GC×GC-TOFMS dataset was analyzed using tile-based Fisher ratio (F-ratio) analysis. Since there were 24 time-point intervals, this constituted 24 sample classes in the F-ratio calculation which produced 672 metabolite hits. Next, application of the computational method determined that there were 210 of the 672 metabolites exhibiting cycling: 55 identified metabolites and 155 unknown metabolites. Furthermore, the 210 cycling metabolites were categorized into four groups, and where applicable, a phase determined: 1 cycle/5 h period (106 metabolites), 2 cycles/5 h period (13 metabolites), spiky pattern (12 metabolites), or multimodal pattern (79 metabolites).

Baseline correction method for dynamic pressure gradient modulated comprehensive two-dimensional gas chromatography with flame ionization detection.

A baseline correction method is developed for comprehensive two-dimensional (2D) chromatography (GC × GC) with flame-ionization detection (FID) using dynamic pressure gradient modulation (DPGM). The DPGM-GC × GC-FID utilized porous layer open tubular (PLOT) columns in both dimensions to focus on light hydrocarbon separations. Since DPGM is nominally a stop-flow modulation technique, a rhythmic baseline disturbance is observed in the FID signal that cycles with the modulation period (PM). This baseline disturbance needs to be corrected to optimize trace analysis. The baseline correction method has three steps: collection of a background "blank" chromatogram and multiplying it by an optimized normalization factor, subtraction of the normalization-optimized background chromatogram from a sample chromatogram, and application of Savitzky-Golay smoothing. An alkane standard solution, containing pentane, hexane and heptane was used for method development, producing linear calibration curves (r2 > 0.991) over a broad concentration range (7.8 ppm - 4000 ppm). Further, the limit-of-detection (LOD) and limit-of-quantification (LOQ) were determined for pentane (LOD = 2.5 ppm, LOQ = 8.2 ppm), hexane (LOD = 0.9 ppm, LOQ = 3.0 ppm), and heptane (LOD = 1.9 ppm, LOQ = 6.4 ppm). A natural gas sample separation illustrated method applicability, whereby the DPGM produced a signal enhancement (SE) of 30 for isopentane, where SE is defined as the height of the tallest 2D peak in the modulated chromatogram for the analyte divided by the height of the unmodulated 1D peak. The 30-fold SE resulted in about a 10-fold improvement in the signal-to-noise ratio (S/N) for isopentane. Additional versatility of the baseline correction method for more complicated samples was demonstrated for an unleaded gasoline sample, which enabled the detection (and visual appearance) of trace components.

Using a 3-hydroxyflavone derivative as a fluorescent probe for the indirect determination of aminothiols separated by ion-pair HPLC.

Homocysteine, cysteine, cysteinyl-glycine, and glutathione are significant biological aminothiols (ATs) that are marker-molecules in Down syndrome, Alzheimer's disease, or have been implicated as risk factors in atherosclerosis and other vascular diseases, and therefore rapid determination of these molecules is desirable. After reduction of the disulfides, a widely used method utilizes derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) as a fluorogenic probe prior to reversed-phase HPLC separation followed by fluorescence detection. The traditional HPLC determination of ATs is time consuming and economically expensive. We have developed an ion-pair HPLC method coupled with indirect fluorescence detection after post-column reaction with a 2,4-dinitrobenzenesulfonate derivative of a 3-hydroxyflavone. The accuracy, precision, post-column temperature and residence time, and limit-of-detection were evaluated. Sample throughput and reduced sample preparation time of over an hour for the existing methods to less than 20 minutes for the new method is also demonstrated. No statistical differences in HCy, Cys, or Cys-Gly determinations in plasma samples were observed between our method and the traditional HPLC method.

Membrane Protein Production in the Yeast, S. cerevisiae.

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca(2+)-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.

The synthesis of recombinant membrane proteins in yeast for structural studies.

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.