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At least 217 records · Page 12

RhizoGrid Indexed Sorghum Rhizosphere Multi-Omics

PerCon SFA project data dentification of spatially resolved biomarkers of drought in Sorghum bicolor rhizosphere molecular-microbe interactions using a novel root cartography "RhizoGrid" system for sampling plants under drought and control conditions across 10 equally sized root zone environments (4 quadrants each). Each quadrant was sampled and processed for 16S amplicon, metabolomics, and X-ray computed tomography (XCT). Data download includes experimental metadata and results files for 16S rRNA sequence analysis of microbial community assembly (processed data files), liquid chromatography mass spectrometry (LC-MS) metabolomics analysis of microbial community root exudates (processed data files), X-ray computed tomography (XCT) spatial gradient analysis (raw and processed data files) of microbial community composition, and related computational modeling outputs.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolome patterns identify active dechlorination in bioaugmentation consortium SDC-9™

Ultra-high performance liquid chromatography–high-resolution mass spectrometry (UPHLC–HRMS) is used to discover and monitor single or sets of biomarkers informing about metabolic processes of interest. The technique can detect 1000’s of molecules (i.e., metabolites) in a single instrument run and provide a measurement of the global metabolome, which could be a fingerprint of activity. Despite the power of this approach, technical challenges have hindered the effective use of metabolomics to interrogate microbial communities implicated in the removal of priority contaminants. Herein, our efforts to circumvent these challenges and apply this emerging systems biology technique to microbiomes relevant for contaminant biodegradation will be discussed. Chlorinated ethenes impact many contaminated sites, and detoxification can be achieved by organohalide-respiring bacteria, a process currently assessed by quantitative gene-centric tools (e.g., quantitative PCR). This laboratory study monitored the metabolome of the SDC-9™ bioaugmentation consortium during cis-1,2-dichloroethene (cDCE) conversion to vinyl chloride (VC) and nontoxic ethene. Untargeted metabolomics using an UHPLC-Orbitrap mass spectrometer and performed on SDC-9™ cultures at different stages of the reductive dechlorination process detected ~10,000 spectral features per sample arising from water-soluble molecules with both known and unknown structures. Multivariate statistical techniques including partial least squares-discriminate analysis (PLSDA) identified patterns of measurable spectral features (peak patterns) that correlated with dechlorination (in)activity, and ANOVA analyses identified 18 potential biomarkers for this process. Statistical clustering of samples with these 18 features identified dechlorination activity more reliably than clustering of samples based only on chlorinated ethene concentration and Dhc 16S rRNA gene abundance data, highlighting the potential value of metabolomic workflows as an innovative site assessment and bioremediation monitoring tool.

environmental monitoring↗

Continuous cropping of potato changed the metabolic pathway of root exudates to drive rhizosphere microflora

For potato production, continuous cropping (CC) could lead to autotoxicity buildup and microflora imbalance in the field soil, which may result in failure of crops and reduction in yield. In this study, non-targeted metabolomics (via liquid chromatography with tandem mass spectrometry (LC–MS/MS)) combined with metagenomic profiling (via high-throughput amplicon sequencing) were used to evaluate correlations between metabolomics of potato root exudates and communities of bacteria and fungi around potato plants to illustrate the impacts of CC. Potato plants were grown in soil collected from fields with various CC years (0, 1, 4, and 7 years). Metabolomic analysis showed that the contents and types of potential autotoxins in potato root exudates increased significantly in CC4 and CC7 plants (i.e., grown in soils with 4 and 7 years of CC). The differentially expressed metabolites were mainly produced via alpha-linolenic acid metabolism in plant groups CC0 and CC1 (i.e., no CC or 1 year CC). The metabolomics of the groups CC4 and CC7 became dominated by styrene degradation, biosynthesis of siderophore group non-ribosomal peptides, phenylpropanoid biosynthesis, and biosynthesis of various plant secondary metabolites. Continuous cropping beyond 4 years significantly changed the bacterial and fungal communities in the soil around the potato crops, with significant reduction of beneficial bacteria and accumulation of harmful fungi. Correlations between DEMs and microflora biomarkers were established with strong significances. These results suggested that continuous cropping of potato crops changed their metabolism as reflected in the plant root exudates and drove rhizosphere microflora to directions less favorable to plant growth, and it needs to be well managed to assure potato yield.

Xing, Yanhong↗

Purification and expression of a novel bacteriocin, JUQZ-1, against Pseudomonas syringae pv. Actinidiae (PSA), secreted by Brevibacillus laterosporus Wq-1, isolated from the rhizosphere soil of healthy kiwifruit

Kiwifruit canker, caused by Pseudomonas syringae pv. actinidiae (PSA), has led to significant losses in the kiwifruit industry each year. Due to the drug resistance feature of PSA, biological control is currently the most promising method. Developing biocontrol bacteria against PSA could help solve the issue of drug resistance generated during the chemical control of PSA to a certain extent. In this research, a Wq-1 strain that demonstrated excellent inhibitory activity against PSA was isolated from the rhizosphere soil of healthy kiwifruit. Based on the morphological characteristics and phylogenetic analysis of the 16S rRNA gene sequence, the isolated strain was identified as Brevibacillus laterosporus Wq-1. Bacteriostatic proteins were isolated from the cell-free culture filtrate of strain Wq-1 and were found to have a molecular weight of approximately 12 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Liquid chromatography–tandem mass spectrometry (LC–MS/MS) detection revealed that there were several peptides in the target band that were consistent with protein 01021 in the genome. The gene of the 01021 protein was cloned into the plasmid pPICZa, and the recombinant bacteriocin was successfully expressed using the Pichia pastoris X33 expression system. The recombinant protein 01021 effectively inhibited the growth of PSA. This is the first report of the protein’s antimicrobial activity, distinguishing it from previously identified bacteriocins. Therefore, we named this bacteriocin JUQZ-1. In addition, our results showed that the protein JUQZ-1 not only exhibited a broad bacteriostatic spectrum but also high thermal and pH stability suitable for harsh environmental conditions., JUQZ-1, a protein with antimicrobial properties and strong environmental tolerance, may serve as a promising alternative to antibiotics.

Shuai, Yang↗

One-Step Regeneration of Hairy Roots to Induce High Tanshinone Plants in Salvia miltiorrhiza

Salvia miltiorrhiza is a traditional Chinese medicinal plant of Labiatae, which has been widely utilized to treat a variety of cardiovascular and cerebrovascular diseases. However, due to the long growth cycle, low content of active ingredients, and serious quality deterioration of S. miltiorrhiza , the use of biotechnology to improve S. miltiorrhiza to meet the growing demand for clinical applications has become a research hotspot. In this study, a novel one-step hairy root regeneration method was developed, which could rapidly obtain hairy roots and regenerated plants with high tanshinone content. By optimizing the parameters of Agrobacterium rhizogenes transformation in S. miltiorrhiza , it was finally established that the explants were infected in Ar.qual (OD 600 = 0.6) for 10 min, co-cultured for 3 days, and then screened on the screening medium containing 7.5 mg/l hygromycin, the maximum transformation frequency can reach 73.85%. GFP and PCR detection yielded a total of 9 positive transgenic hairy root lines and 11 positive transgenic regenerated plants. SmGGPPS1 was successfully overexpressed in positive transgenic regenerated plants, according to the results of qRT-PCR. The content of tanshinone IIA and cryptotanshinone were dramatically enhanced in transgenic regenerated plants and hairy roots by Ultra Performance Liquid Chromatography analysis. Based on the Agrobacterium-mediated transformation of S. miltiorrhiza , this study developed a new method for regenerating plants with transgenic hairy roots. This method provides a foundation for the breeding of S. miltiorrhiza and the sustainable development of medicinal plant resources, as well as provides a useful reference for the application of other species.

Su, Yuekai↗

Discovery top-down proteomics in symbiotic soybean root nodules

Proteomic methods have been widely used to study proteins in complex biological samples to understand biological molecular mechanisms. Most well-established methods (known as bottom-up proteomics, BUP) employ an enzymatic digestion step to cleave intact proteins into smaller peptides for liquid chromatography (LC) mass spectrometry (MS) detection. In contrast, top-down proteomics (TDP) directly characterizes intact proteins including all possible post-translational modifications (PTMs), thus offering unique insights into proteoform biology where combinations of individual PTMs may play important roles. We performed TDP on soybean root nodules infected by the symbiotic Bradyrhizobium japonicum in both the wildtype bacterium and a nifH- mutant, which lacks the ability to fix nitrogen in the soybean root nodule. TDP captured 1648 proteoforms derived from 313 bacterial genes and 178 soybean genes. Leghemoglobin, the most abundant protein in the sample, existed in many truncated proteoforms. Interestingly, these truncated proteoforms were considerably more abundant in the wildtype relative to the nifH- mutant, implicating protease activity as an important factor in nitrogen fixation. Proteoforms with various PTMs and combinations thereof were identified using an unrestricted open modification search. This included less common PTMs such as myristoylation, palmitoylation, cyanylation, and sulfation. In parallel, we collected high resolution MS imaging (MSI) data of intact proteins and biopolymers (<20 kDa due to current technical limitations) from sections of the soybean root nodules using matrix-assisted laser desorption/ionization (MALDI) coupled to high resolution Orbitrap. Several detected proteoforms exhibited unique spatial distributions inside the infection zone and cortex, suggesting functional compartmentalization in these regions. A subset of peaks from the MALDI-MSI were assigned to proteoforms detected in TDP LCMS data based on matching accurate masses. Many of the proteins detected in both LCMS and MALDI-MSI are currently uncharacterized in UniProt: the PTM and spatial information presented here will be valuable in understanding their biological functions. Taken together, our study demonstrates how untargeted TDP approach can provide unique insights into plant proteoform biology. On-going technology developments are expected to further improve TDP coverage for more comprehensive high-throughput analysis of proteoforms.

59 BASIC BIOLOGICAL SCIENCES↗

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

59 BASIC BIOLOGICAL SCIENCES↗

Sensing Techniques for Organochlorides through Intermolecular Interaction with Bicyclic Amidines

Toxic organochloride molecules are widely used in industry for various purposes. With their high volatility, the direct detection of organochlorides in environmental samples is challenging. Here, a new organochloride detection mechanism using 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) is introduced to simplify a sensing method with higher detection sensitivity. Three types of organochloride compounds-trichloroethylene (TCE), dichloromethane (DCM), and dichlorodiphenyltrichloroethane (DDT)—were targeted to understand DCM conjugation chemistry by using nuclear magnetic resonance (NMR) and liquid chromatography with a mass spectrometer (LC-MS). 13C-NMR spectra and LC-MS data indicated that DBN can be labeled on these organochloride compounds by chlorine–nitrogen interaction. Furthermore, to demonstrate the organochloride sensing capability, the labeling yield and limit of detection were determined by a colorimetric assay as well as micellar electrokinetic chromatography (MEKC). The interaction with DBN was most appreciable for TCE, among other organochlorides. TCE was detected at picomolar levels, which is two orders of magnitude lower than the maximum contaminant level set by the United States Environmental Protection Agency. MEKC, in conjunction with this DBN-labeling method, enables us to develop a field-deployable sensing platform for detecting toxic organochlorides with high sensitivity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Evaluating Software Tools for Lipid Identification from Ion Mobility Spectrometry–Mass Spectrometry Lipidomics Data

The unambiguous identification of lipids is a critical component of lipidomics studies and greatly impacts the interpretation and significance of analyses as well as the ultimate biological understandings derived from measurements. The level of structural detail that is available for lipid identifications is largely determined by the analytical platform being used. Mass spectrometry (MS) coupled with liquid chromatography (LC) is the predominant combination of analytical techniques used for lipidomics studies, and these methods can provide fairly detailed lipid identification. More recently, ion mobility spectrometry (IMS) has begun to see greater adoption in lipidomics studies thanks to the additional dimension of separation that it provides and the added structural information that can support lipid identification. At present, relatively few software tools are available for IMS-MS lipidomics data analysis, which reflects the still limited adoption of IMS as well as the limited software support. This fact is even more pronounced for isomer identifications, such as the determination of double bond positions or integration with MS-based imaging. In this review, we survey the landscape of software tools that are available for the analysis of IMS-MS-based lipidomics data and we evaluate lipid identifications produced by these tools using open-access data sourced from the peer-reviewed lipidomics literature.

59 BASIC BIOLOGICAL SCIENCES↗

Chemical Characterization and Antimicrobial Activity of Green Propolis from the Brazilian Caatinga Biome

Green propolis, particularly from the unique flora of the Brazilian Caatinga biome, has gained significant interest due to its diverse chemical composition and biological activities. This study focuses on the chemical characterization and antimicrobial evaluation of Caatinga green propolis. Twelve compounds were isolated through different chromatographic techniques, including flavanones (naringenin, 7-O-methyleriodictyol, sakuranetin), flavones (hispidulin, cirsimaritin), flavonols (quercetin, quercetin-3-methyl ether, kaempferol, 6-methoxykaempferol, viscosine, penduletin), and one chalcone (kukulkanin B). Using liquid chromatography–quadrupole time-of-flight tandem mass spectrometry (LC-QToF-MS), a total of 55 compounds excluding reference standards were tentatively identified, which include flavonoids, phenolic acids derivatives, and alkaloids, with flavonols, flavanones, and flavones being predominant. Antimicrobial testing against pathogens revealed that the crude extract exhibited low inhibitory activity, against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) (IC50: 148.4 and 120.98 µg/mL, respectively). Although the isolated compounds showed limited individual activity, a fraction containing sakuranetin and penduletin (Fraction 8) exhibited moderated activity against Cryptococcus neoformans (IC50: 47.86 µg/mL), while a fraction containing quercetin and hispidulin showed moderated activity against VRE (IC50: 16.99 µg/mL). These findings highlight the potential application of Caatinga green propolis as an antimicrobial agent, particularly against resistant bacterial strains, and underscore the importance of synergistic interactions between compounds in enhancing biological effects.

Aldana-Mejía, Jennyfer A. (ORCID:0000000307361899)↗

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors including histone PTMs are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for engineering solutions to increase the resilience of crops to climate change. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed as their intact forms by top-down mass spectrometry (MS) coupled to online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow thus yielding the global PTM profile of core histones (H4, H2A, H2B, H3). By comparing the PTM profile among samples corresponding to different conditions (e.g. drought vs. control), potential epigenetic marks can be discovered as targets for further characterization using approaches such as chromatin immunoprecipitation – sequencing (ChIP-seq).

59 BASIC BIOLOGICAL SCIENCES↗

Carrier-assisted One-pot Sample Preparation for Targeted Proteomics Analysis of Small Numbers of Human Cells

Protein analysis of small numbers of human cells is primarily achieved by targeted proteomics with antibody-based immunoassays, which have inherent limitations (e.g., low multiplex and unavailability of antibodies for new proteins). Mass spectrometry (MS)-based targeted proteomics has emerged as an alternative because it is antibody-free, high multiplex, and has high specificity and quantitation accuracy. Recent advances in MS instrumentation make MS-based targeted proteomics possible for multiplexed quantification of highly abundant proteins in single cells. However, there is a technical challenge for effective processing of single cells with minimal sample loss for MS analysis. To address this issue, we have recently developed a convenient protein carrier-assisted one-pot sample preparation coupled with liquid chromatography (LC) - selected reaction monitoring (SRM) termed cLC-SRM for targeted proteomics analysis of small numbers of human cells. This method capitalizes on using the combined excessive exogenous protein as a carrier and low-volume one-pot processing to greatly reduce surface adsorption losses as well as high-specificity LC-SRM to effectively address the increased dynamic concentration range due to the addition of exogeneous carrier protein. Its utility has been demonstrated by accurate quantification of most moderately abundant proteins in small numbers of cells (e.g., 10-100 cells) and highly abundant proteins in single cells. The easy-to-implement features and no need for specific devices make this method readily accessible to most proteomics laboratories. In this study, we have provided a detailed protocol for cLC-SRM analysis of small numbers of human cells including cell sorting, cell lysis and digestion, LC-SRM analysis, and data analysis. Further improvements in detection sensitivity and sample throughput are needed towards targeted single-cell proteomics analysis. We anticipate that cLC-SRM will be broadly applied to biomedical research and systems biology with the potential of facilitating precision medicine.

60 APPLIED LIFE SCIENCES↗

Meta-analysis of peptides to detect protein significance

Shotgun assays are widely used in biotechnologies to char- acterize large molecules, which are hard to be measured as a whole directly. For instance, in Liquid Chromatography Mass Spectrometry (LC-MS) shotgun experiments, proteins in biological samples are digested into peptides, and then peptides are separated and measured. However, in proteomics study, investigators are usually interested in the performance of the whole proteins instead of those peptide fragments. In light of meta-analysis, we propose an adaptive thresholding method to select informative peptides, and combine peptide-level models to protein-level analysis. Overall, the meta-analysis procedure and modeling rationale can be adapted to data analysis of other types of shotgun assays.

59 BASIC BIOLOGICAL SCIENCES↗

Seasonal investigation of ultrafine-particle organic composition in an eastern Amazonian rainforest

Abstract. Reports on the composition of ultrafine particles (<100 nm in diameter) in the Amazon are scarce, due in part to the fact that new-particle formation has rarely been observed near ground level. Ultrafine particles near the surface have nevertheless been observed, leaving open questions regarding the sources and chemistry of their formation and growth, particularly as these vary across seasons. Here, we present measurements of the composition of ultrafine particles collected in the Tapajós National Forest (2.857° S, 54.959° W) during three different seasonal periods: 10–30 September 2016 (SEP), 18 November–23 December 2016 (DEC), and 22 May–21 June 2017 (JUN). Size-selected (5–70 nm) particles were collected daily (for 22 h each day) using an offline sampler. Samples collected during the three time periods were compiled and analyzed using liquid chromatography coupled with Orbitrap high-resolution mass spectrometry. Our findings suggest a sustained influence of isoprene organosulfate chemistry on ultrafine particles from the different periods. We present chemical evidence that indicates that biological-spore fragmentation impacted ultrafine-particle composition during the late wet season (JUN), while chemical markers for biomass burning and secondary chemistry peaked during the dry season (SEP and DEC). Higher oxidation states and degrees of unsaturation were observed for organics in the dry season (SEP and DEC), suggesting greater extents of aerosol aging. Finally, applying a volatility parameterization to the observed compounds suggests that organic sulfur species are likely key drivers of new-particle growth in the region due to their low volatility compared to other species.

54 ENVIRONMENTAL SCIENCES↗

Ice nucleation measurements from DRUM impactors during TRACER campaign in the Houston TX region from July to September 2022

During TRACER, three Davis Rotating Uniform size-cut Monitors (DRUM; DRUMAir 4-DRUM) were used to collect aerosols for ice nucleation measurements in the Brooks laboratory at Texas A&M University. The three instruments were located at AMF1 in La Porte, Ancillary site in Guy (ANC), and onboard the Texas A&M Rapid Onsite Atmospheric Measurements Van (ROAM-V). ROAM-V was deployed to capture airmasses behind (maritime) and ahead (continental) of the passage of the sea-breeze front through Houston. On select sampling days, ROAM-V sampled in the morning/mid-day on the coast and then transited to a second inland site for the afternoon/evening. The suite of instruments deployed on ROAM-V included a Condensation Particle Counter (CPC; GRIMM Model 5.403 CPC), Scanning Mobility Particle Sizer (SMPS; TSI 3750 detector, TSI 3082 classifier, TSI 3088 neutralizer, TSI 3081A Differential Mobility Analyzer), Cloud Condensation Nuclei counter (Droplet Measurement Technologies CCN Counter), micro pulse lidar (Droplet Measurement Technologies Micro Pulse LiDAR (miniMPL)), and one of the Davis Rotating Uniform size-cut Monitors (DRUM; DRUMAir 4-DRUM). Before sampling at each location, the latitude and longitude were recorded using the GPS on the phone application “My Altitude”. Each DRUM sampler was operated at a flow rate of 23 LPM. Each DRUM has four stages where the aerodynamic diameter size cuts are as follows: stage 1 larger than 3 μm, stage 2 from 3 to 1.2 μm, stage 3 from 1.2 to 0.34 μm, and stage 4 from 0.34 to 0.15 μm. Pretreated aluminum foil was used as a substrate on all stages. At AMF1 and ANC sites, the DRUMs were operated on the shared aerosol inlet, with generous support of the DOE ARM site staff. These instruments rotated 24 mm per day and only contained aluminum foil substrates. Substrates were changed weekly and transported to Texas A&M for storage in -80C freezer until analysis. The DRUM onboard ROAM-V was operated at a faster rotation rate of 150 mm per day to clearly separate the multiple deployment locations for ROAM-V. For the ROAM-V DRUM, substrates were changed every deployment and transported to Texas A&M for storage in -80C freezer until analysis. At Texas A&M, ice nucleation experiments were conducted to measure the ice nucleation temperature of the ambient aerosol samples collected from the three DRUMs using our previously established procedures (Alsante et al., 2023; Fornea et al., 2009; Matthews et al., 2023). For ice nucleation, only samples collected on stage 3 were analyzed, given that these are the most relevant size (1.2 to 0.34 μm diameter) for potential ice nucleating particles. For the AMF1 and ANC sites, we cut and analyzed 2 mm (2-hour) samples. We analyzed the time periods of the AMF1 site instrument when the ROAM-V was deployed. A 72-hour period from July 11th at 23:49 through July 15th at 1:49 was analyzed from the ANC site instrument. For the ROAM-V instrument, we separated the daily samples by site location. Between 1- and 6-hour independent samples were analyzed at each location. We also cut the ROAM-V samples in half to allow for compositional analysis of the aerosol on the other half of the substrate. All viable samples from the ROAM-V were analyzed. Analysis was done using a custom-built ice nucleation apparatus, recently updated to include an array of 16 individual samples (Matthews et al., 2023). On our experimental setup, we used 100 μL of Ultra-High-Performance Liquid Chromatography (UHPLC) water (Sigma Aldrich, >99.9% purity) to wash off the aerosol from the DRUM substrate. Then, we micropipetted 2 μL droplet samples into each of the 16 wells of the array, using hydrophobically coated microscope slides. Experiments would cycle 28 times from 10 C to -40C over a 20-hour period. During analysis of the TRACER samples, nine experiments were conducted with UHPLC water process blanks that followed an identical preparation procedure. The average freezing temperature and standard deviation for these process blanks is -27.7±2.4 C. This data was collected for ARM Field Campaign AFC07023 and supported by DOE ASR grant DE-SC0021047. For any further questions, please feel free to contact the instrument PI, Sarah D. Brooks, sbrooks@tamu.edu. Alsante, A. N., Thornton, D. C., & Brooks, S. D. (2023). Ice nucleation catalyzed by the photosynthesis enzyme RuBisCO and other abundant biomolecules. Communications Earth & Environment, 4(1), 51. Fornea, A. P., Brooks, S. D., Dooley, J. B., & Saha, A. (2009). Heterogeneous freezing of ice on atmospheric aerosols containing ash, soot, and soil. Journal of Geophysical Research: Atmospheres, 114(D13). DOI:10.1029/2009JD011958 Matthews, B. H., Alsante, A. N., & Brooks, S. D. (2023). Pollen Emissions of Subpollen Particles and Ice Nucleating Particles. ACS Earth and Space Chemistry.

54 ENVIRONMENTAL SCIENCES↗

Characterizing intracellular proteomes for microbes: An experimental approach using label-free protein quantitation

Genomic and transcriptomic studies generate a rich source of molecular information; however, neither a static genome nor the presence of a messenger RNA provides the most direct insight into the functional state of a microbial cell at any given time. Rather, it is the ensemble of proteins (i.e., proteome) that is the primary workhorse for most biological processes that are concurrent and coordinately active in a living cell. Currently, mass-spectrometry-based (MS) technologies provide unprecedented information into the composition of the proteome, shedding light on the numerous complex biological processes actively shaping observed phenotypes. Herein, we detail a protocol for the exploration of intracellular proteomes through the large-scale, unbiased identification of proteins and their relative abundances using liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). In general, this protocol is applicable to both microbial and eukaryotic systems.

Abraham, Paul↗

Mass spectrometry-based proteomics for analysis of hydrophilic phosphopeptides

Protein phosphorylation is a critical post-translational modification (PTM), with cell signaling networks being tightly regulated by protein phosphorylation. Despite recent technological advances in reversed-phase liquid chromatography (RPLC)-mass spectrometry (MS)-based proteomics, comprehensive phosphoproteomic coverage in complex biological systems remains challenging, especially for hydrophilic phosphopeptides that often have multiple phosphorylation sites. Herein we describe an MS-based phosphoproteomics protocol for effective quantitative analysis of hydrophilic phosphopeptides. This protocol was built upon a simple tandem mass tag (TMT)-labeling method for significantly increasing peptide hydrophobicity, thus effectively enhancing RPLC-MS analysis of hydrophilic peptides. Through phosphoproteomic analyses of MCF7 cells, this method was demonstrated to greatly increase the number of identified hydrophilic phosphopeptides and improve MS signal detection. With the TMT labeling method, we were able to identify a previously unreported phosphopeptide from the G protein-coupled receptor (GPCR) CXCR3, QPpSSSR, which is thought to be important in regulating receptor signaling. This protocol is easy to adopt and implement, and thus should have broad utility for effective RPLC-MS analysis of the hydrophilic phosphoproteome as well as other highly hydrophilic analytes.

Hydrophilic phosphopeptide, Phosphoproteomics, TMT↗

Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.

mass spectrometry↗