Atomic Force Microscope Controlled Topographical Imaging and Proximal Probe Thermal Desorption/Ionization Mass Spectrometry Imaging

This paper reports on the development of a hybrid atmospheric pressure atomic force microscopy/mass spectrometry imaging system utilizing nanothermal analysis probes for thermal desorption surface sampling with subsequent atmospheric pressure chemical ionization and mass analysis. The basic instrumental setup and the general operation of the system were discussed, and optimized performance metrics were presented. The ability to correlate topographic images of a surface with atomic force microscopy and a mass spectral chemical image of the same surface, utilizing the same probe without moving the sample from the system, was demonstrated. Co-registered mass spectral chemical images and atomic force microscopy topographical images were obtained from inked patterns on paper as well as from a living bacterial colony on an agar gel. Spatial resolution of the topography images based on pixel size (0.2 μm × 0.8 μm) was better than the resolution of the mass spectral images (2.5 μm × 2.0 μm), which were limited by current mass spectral data acquisition rate and system detection levels

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Characterization of Indole-3-acetic Acid Biosynthesis and the Effects of This Phytohormone on the Proteome of the Plant-Associated Microbe <i>Pantoea</i> sp. YR343

Indole-3-acetic acid (IAA) plays a central role in plant growth and development, and many plant-associated microbes produce IAA using tryptophan as the precursor. Using genomic analyses, we predicted that <i>Pantoea</i> sp. YR343, a microbe isolated from <i>Populus deltoides</i>, synthesizes IAA using the indole-3-pyruvate (IPA) pathway. To better understand IAA biosynthesis and the effects of IAA exposure on cell physiology, we characterized proteomes of <i>Pantoea</i> sp. YR343 grown in the presence of tryptophan or IAA. Exposure to IAA resulted in upregulation of proteins predicted to function in carbohydrate and amino acid transport and exopolysaccharide (EPS) biosynthesis. Metabolite profiles of wild-type cells showed the production of IPA, IAA, and tryptophol, consistent with an active IPA pathway. Finally, we constructed an Δ<i>ipdC</i> mutant that showed the elimination of tryptophol, consistent with a loss of IpdC activity, but was still able to produce IAA (20% of wild-type levels). Although we failed to detect intermediates from other known IAA biosynthetic pathways, this result suggests the possibility of an alternate pathway or the production of IAA by a nonenzymatic route in <i>Pantoea</i> sp. YR343. The Δ<i>ipdC</i> mutant was able to efficiently colonize poplar, suggesting that an active IPA pathway is not required for plant association

Proteomics-Based Tools for Evaluation of Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) has the potential to produce enzymes, therapeutic agents, and other proteins, while circumventing difficulties associated with in vivo heterologous expression. However, the contents of the cell-free extracts used to carry out synthesis are generally not characterized, which hampers progress toward enhancing yield or functional activity of the target protein. We explored the utility of mass spectrometry (MS)-based proteomics for characterizing the bacterial extracts used for transcribing and translating gene sequences into proteins as well as the products of CFPS reactions. Full proteome experiments identified over 1000 proteins per reaction. The complete set of proteins necessary for transcription and translation were found, demonstrating the ability to define potential metabolic capabilities of the extract. Further, MS-based techniques allowed characterization of the CFPS product and provided insight into the synthesis reaction and potential functional activity of the product. These capabilities were demonstrated using two different CFPS products, the commonly used standard green fluorescent protein (GFP, 27 kDa) and the polyketide synthase DEBS1 (394 kDa). For the large, multidomain DEBS1, substantial premature termination of protein translation was observed. Additionally, MS/MS analysis, as part of a conventional full proteomics workflow, identified post-translational modifications, including the chromophore in GFP, as well as the three phosphopantetheinylation sites in DEBS1. A hypothesis-driven approach focused on these three sites identified that all were correctly modified for DEBS1 expressed in vivo but with less complete coverage for protein expressed in CFPS reactions. These post-translational modifications are essential for functional activity, and the ability to identify them with mass spectrometry is valuable for judging the success of the CFPS reaction. Collectively, the use of MS-based proteomics will prove advantageous for advancing the application of CFPS and related techniques

Comparison of array data to significantly different proteins.

a<p>Ratios are represented as log2 values for 10 h treatment condition over 6 h control condition.</p>b<p>Significance index shows if a gene was significantly differentially expressed (1) or not (0) for this comparison.</p

The growth, glucose consumption and net ethanol production of <i>Z. mobilis</i> in the absence or presence of 47 g/L (or 6% [v/v]) ethanol.

<p>Black and grey arrows indicate the sampling time points of transcriptomic and metabolomic studies for <i>Z. mobilis</i> in the absence of ethanol (control, black arrows) or in the presence of ethanol (treatment, grey arrows) respectively. The exponential phase samples (control at 6 h and treatment at 10 h post-inoculation) were used for proteomic study.</p

Correlations between log₂ based expression ratios from transcriptomic and proteomic studies at exponential phase (ethanol-treated cell at 10 h versus control cells at 6 h).

a<p><b>:</b> The direction of comparison; <b>1</b>: from significant protein list to identify their corresponding genes for correlation; <b>2</b>: from significant gene list to identify their corresponding proteins for correlation; <b>3</b>: both the proteins and their corresponding genes used for correlation calculation have same statistical significant differential expression level, and the results of comparisons are same from either directions. <b>P</b>: proteomics, <b>A</b>: transcriptomics; <b>S</b>: statistically significant, <b>1.5</b>: at least 1.5-fold difference; <b>2.0</b>: at least 2-fold difference. The numbers for proteomic and transcriptomic studies before comparison are: <b>P</b>: 942; <b>SP</b>: 95 (94 proteins with corresponding gene expression were used for <b>SP</b>); <b>SP1.5</b>: 84; <b>SP2.0</b>: 61; <b>A</b>: 1694; <b>SA</b>: 912; <b>SA1.5</b>: 174; <b>SA2.0</b>: 48. <b>Number</b>: the number of gene-protein pairs after comparison; <b>Correlation</b>: the R-squared number between the log2 based expression ratio (ethanol-treated cell versus control cells) of proteins identified from proteomics and log2 based expression ratio (ethanol-treated cell versus control cells) of genes identified from microarray. <b>→:</b> the direction for comparison; For example, <b>P→A</b> is to identify the corresponding genes in microarray data from the protein list of proteomic data. <b>SP1.5→A</b> is to identify the corresponding genes in microarray data from the protein list of proteomic data with at least 1.5-fold changes.</p

Systems Biology Analysis of <i>Zymomonas mobilis</i> ZM4 Ethanol Stress Responses

<div><p>Background</p><p><i>Zymomonas mobilis</i> ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully.</p><p>Methodology/Principal Findings</p><p>In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced <i>Z. mobilis</i> ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update <i>Z. mobilis</i> ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher.</p><p>Conclusions</p><p>Our study has provided insights into the responses of <i>Z. mobilis</i> to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key <i>Z. mobilis</i> ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress.</p></div