Optimized sample buffer for dispersed, high-resolution capillary zone electrophoretic separation of Escherichia coli B.

Capillary zone electrophoresis (CZE) is a powerful tool for high resolution chemical separations. Applying CZE to microbial samples may facilitate a deeper understanding of bacterial physiology and behavior. However, the study of complex microbial samples has been limited by the uncontrolled hetero-aggregation of bacterial cells under an applied electric field. We tested a wide range of sample buffers and buffer additives for the optimization of bacterial CZE separations using a 20 mM Tris-HCl background electrolyte. By modifying the sample buffer, but not the background electrolyte, we retain constant separation conditions, which aids in the comparison of the sample buffer additives. We report optimized methods for automated CZE separation and simultaneous fractionation of Escherichia coli B, which is one of the two most widely used wild-type strains. A modified sample buffer containing neutral salts and the addition of glycerol produced a 20-fold increase in loading capacity and a reduction in peak width/broadening of 86% in comparison to previously reported work. In addition, the glycerol-modified sample buffer appears to reduce the persistent aggregation and adhesion to the capillary walls during electrophoretic separations of complex environmental microbiota.

Comparison of RT-dPCR and RT-qPCR and the effects of freeze-thaw cycle and glycine release buffer for wastewater SARS-CoV-2 analysis.

Public health efforts to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rely on accurate information on the spread of the disease in the community. Acute and surveillance testing has been primarily used to characterize the extent of the disease. However, obtaining a representative sample of the human population is challenging because of limited testing capacity and incomplete testing compliance. Wastewater-based epidemiology is an agnostic alternative to surveillance testing that provides an average sample from the population served by the treatment facility. We compare the performance of reverse transcription quantitative PCR (RT-qPCR) and reverse transcription digital droplet PCR (RT-dPCR) for analysis of SARS-CoV-2 RNA in a regional wastewater treatment facility in northern Indiana, USA from the earliest stages of the pandemic. 1-L grab samples of wastewater were clarified and concentrated. Nucleic acids were extracted from aliquots and analyzed in parallel using the two methods. Synthetic viral nucleic acids were used for method development and generation of add-in standard-curves. Both methods were highly sensitive in detecting SARS-CoV-2 in wastewater, with detection limits as low as 1 copy per 500 mL wastewater. RT-qPCR and RT-dPCR provided essentially identical coefficients of variation (s/[Formula: see text] = 0.15) for triplicate measurements made on wastewater samples taken on 16 days. We also observed a sevenfold decrease in viral load from a grab sample that was frozen at - 80 °C for 92 days compared to results obtained without freezing. Freezing samples before analysis should be discouraged. Finally, we found that treatment with a glycine release buffer resulted in a fourfold inhibition in RT-qPCR signal; treatment with a glycine release buffer also should be discouraged. Despite their prevalence and convenience in wastewater analysis, glycine release and freezing samples severely and additively (~ tenfold) degraded recovery and detection of SARS-CoV-2.

Seamlessly Integrated Miniaturized Filter-Aided Sample Preparation Method to Fractionation Techniques for Fast, Loss-Less, and In-Depth Proteomics Analysis of 1 µg of Cell Lysates at Low Cost.

We report an integrated platform that enabled a seamlessly coupling miniaturized filter-aided sample preparation (MICROFASP) method to high-pH reversed phase (RP) or strong cation exchange (SCX) microreactors for low-loss sample preparation and fractionation of 1 µg of cell lysates prior to LC-ESI-MS/MS analysis. Due to the reduced size of the microreactor, only 5 µL of buffer volume is required to generate each fraction, which speeds both elution and lyophilization. The fraction was directly eluted into an autosampler insert vial for LC-MS analysis to reduce sample transfer steps and minimize sample loss as well as contamination. The flow-through sample generated during the loading step was also collected and analyzed. The integrated platform generated 48,890 unique peptides and 4723 protein groups from 1 µg of a K562 cell lysate using MICROFASP and C18 microreactor-based high-pH RP fractionation methods, which are comparable with the state-of-the-art result using in-StageTip sample preparation and nanoflow RPLC-based fractionation methods but with a significant reduction in cost and time. Both pH gradient elution and salt gradient elution approaches provide high reproducibility for the SCX microreactor-based fractionation method. This integrated platform has significant potential in deep proteomics analysis of mass-limited samples with reduced time and equipment requirements.

3-D printed injection system for capillary electrophoresis.

Commercial systems for capillary electrophoresis are designed for the unattended analysis of several samples, and are usually large, complex, and expensive. We report a compact system for manual injection of a single sample in capillary electrophoresis, which is ideal for method development and for student training. The injector consists of two parts that are manufactured by three-dimensional printing (STL and STEP files are included as ESI). One part is immobile and holds an electrode for powering electrophoresis and a gas line for pressurized injection and pumping fluids through the capillary. The second part is removable and is used to hold washing solutions, separation electrolyte, or sample. Conventional machining is used to tap holes to hold the electrode, separation capillary, gas line, and safety interlock. The system is used for either pressure or electrokinetic sample injection, and can be used to pump fluids through the capillary for changing background electrolytes and reconditioning the capillary between runs. We coupled the injection system to our high-dynamic range laser-induced fluorescence detector and evaluated the system by performing capillary zone electrophoresis on solutions of fluorescein. Electrokinetic injection produced a linear response across five orders of magnitude dynamic range (slope of the log-log calibration curve was 1.02), concentration detection limits of 5 pM, and mass detection limits of 1 zmol. Pressure injection produced a linear response across at least four orders of magnitude (slope of the log-log calibration curve was 0.92), concentration detection limits of 2 pM, and mass detection limits of 10 zmol.

High-Throughput, Comprehensive Single-Cell Proteomic Analysis of Xenopus laevis Embryos at the 50-Cell Stage Using a Microplate-Based MICROFASP System.

We report both the design of a high-throughput MICROFASP (a miniaturized filter aided sample preparation) system and its use for the comprehensive proteomic analysis of single blastomeres isolated from 50-cell stage Xenopus laevis embryos (∼200 ng of yolk-free protein/blastomere). A single run of the MICROFASP system was used to process 146 of these blastomeres in parallel. Three samples failed to generate signals presumably due to membrane clogging. Two cells were lost due to operator error. Of the surviving samples, 32 were analyzed using a Q Exactive HF mass spectrometer in survey experiments (data not included). The 109 remaining blastomeres were analyzed using a capillary LC-ESI-MS/MS system coupled to an Orbitrap Fusion Lumos mass spectrometer, which identified a total of 4189 protein groups and 40,998 unique peptides. On average, 3468 ± 229 protein groups and 14,525 ± 2437 unique peptides were identified from each blastomere, which is the highest throughput and deepest proteome coverage to date of single blastomeres at this stage of development. We also compared two dissociation buffers, Newport and calcium-magnesium-free (CMFM) buffers; the two buffers generated similar numbers of protein identifications (3615 total protein IDs from use of the Newport dissociation buffer and 3671 total protein IDs from use of the CMFM buffer).

Aquaporin-7 Regulates the Response to Cellular Stress in Breast Cancer.

The complex yet interrelated connections between cancer metabolism, gene expression, and oncogenic driver genes have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. Here we effectively integrated metabolomics and gene expression data from breast cancer mouse models through a novel unbiased correlation-based network analysis. This approach identified 35 metabolite and 34 gene hubs with the most network correlations. These hubs have prognostic value and are likely integral to tumor metabolism and breast cancer. The gene hub Aquaporin-7 (Aqp7), a water and glycerol channel, was identified as a novel regulator of breast cancer. AQP7 was prognostic of overall survival in patients with breast cancer. In mouse breast cancer models, reduced expression of Aqp7 caused reduced primary tumor burden and lung metastasis. Metabolomics and complex lipid profiling of cells and tumors with reduced Aqp7 revealed significantly altered lipid metabolism, glutathione metabolism, and urea/arginine metabolism compared with controls. These data identify AQP7 as a critical regulator of metabolic and signaling responses to environmental cellular stresses in breast cancer, highlighting AQP7 as a potential cancer-specific therapeutic vulnerability. SIGNIFICANCE: Aquaporin-7 is identified as a critical regulator of nutrient availability and signaling that responds to cellular stresses, making it an attractive therapeutic target in breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4071/F1.large.jpg.

Minimal deuterium isotope effects in quantitation of dimethyl-labeled complex proteomes analyzed with capillary zone electrophoresis/mass spectrometry.

Stable heavy-isotope labeling is commonly used in quantitative proteomics. Several common techniques incorporate deuterium (2 H) as the heavy isotopic label using reductive amination with formaldehyde. Compared with alternatives, dimethyl labeling reagents are inexpensive and the labeling chemistry is simple and rapid. However, the substitution of hydrogen by deuterium can introduce subtle changes in peptides' polarities, leading to a shift in chromatographic retention times between deuterated and nondeuterated peptides that can lead to quantification deviations. Capillary zone electrophoresis has emerged as a complementary separation for ESI-MS-based proteomics, including targeted and quantitative approaches. The extent to which the deuterium isotope effect impacts CZE-based proteomics, which separates peptides based on their S/N ratios, has not been investigated. To address this issue, CZE was used to analyze dimethyl labeled E. coli tryptic digests in 100 min single-shot analyses. The median migration time shift was 0.1 s for light versus heavy labeled peptides, which is 2.5% of the peak width. For comparison, nUHPLC-ESI-MS/MS was used to analyze the same sample. In UPLC, deuterated peptides tended to elute earlier than nondeuterated peptides, with a retention shift of 3 s for light versus heavy labeled peptides, which is roughly half the peak width. This shift in separation time did not have a significant effect on quantitation for either method for equal mixing ratios of the light-intermediate-heavy isotope labeled samples.

Capillary zone electrophoresis separation and collection of spermatozoa for the forensic analysis of sexual assault evidence.

The processing of sexual assault kits (SAKs) relies on the genetic analysis of material extracted from swabs collected from the assault victim. A vital step in producing an identifiable DNA profile of the perpetrator is the effective separation of perpetrator (sperm) and victim (epithelial) DNA that have been isolated from the collected evidence. We report the use of capillary zone electrophoresis for the separation of intact sperm from whole and lysed epithelial cells in SAKs. The separated components are deposited into wells of a microtiter plate using a computer-controlled fraction collector, and quantitative PCR is used to verify the collection of sperm cells by targeted amplification of male DNA. We present results from simulated sexual assault samples that have been aged for up to 18 months, as well as vaginal swabs from authentic forensic kits. Components extracted from the vaginal swabs from the SAK comigrated with an aged semen sample at 6.25 ± 0.25 min. Epithelial cells migrated from 10-12 min, producing baseline resolution of the components. Sperm cells were collected in a microtiter plate for downstream analysis.

Miniaturized Filter-Aided Sample Preparation (MICRO-FASP) Method for High Throughput, Ultrasensitive Proteomics Sample Preparation Reveals Proteome Asymmetry in Xenopus laevis Embryos.

We report a miniaturized filter aided sample preparation method (micro-FASP) for low-loss preparation of submicrogram proteomic samples. The method employs a filter with ∼0.1 mm2 surface area, reduces the total volume of reagents to <10 µL, and requires only two sample transfer steps. The method was used to generate 25 883 unique peptides and 3069 protein groups from 1000 MCF-7 cells (∼100 ng protein content), and 13 367 peptides and 1895 protein groups were identified from 100 MCF-7 cells (∼10 ng protein content). Single blastomeres from Xenopus laevis embryos at the 50-cell stage (∼200 ng yolk free protein/blastomere) generated 20 943 unique peptides and 2597 protein groups; the proteomic profile clearly differentiated left and right blastomeres and provides strong support for models in which this asymmetry is established early in the embryo. The parallel processing of 12 samples demonstrates reproducible label free quantitation of 1 µg protein homogenates.

Quantitative capillary zone electrophoresis-mass spectrometry reveals the N-glycome developmental plan during vertebrate embryogenesis.

Glycans are known to be involved in many biological processes, while little is known about the expression of N-glycans during vertebrate development. We now report the first quantitative studies of both the expression of N-linked glycans at six early development stages and the expression of N-glycosylated peptides at two early development stages in Xenopus laevis, the African clawed frog. N-Glycans were labeled with isobaric tandem mass tags, pooled, separated by capillary electrophoresis, and characterized using tandem mass spectrometry. We quantified 110 N-glycan compositions that spanned four orders of magnitude in abundance. Capillary electrophoresis was particularly useful in identifying charged glycans; over 40% of the observed glycan compositions were sialylated. The glycan expression was relatively constant until the gastrula-neurula transition (developmental stage 13), followed by massive reprogramming. An increase in oligomannosidic and a decrease in the paucimannosidic and phosphorylated oligomannosidic glycans were observed at the late tailbud stage (developmental stage 41). Two notable and opposing regulation events were detected for sialylated glycans. LacdiNAc and Lewis antigen features distinguished down-regulated sialylation from up-regulated species. The level of Lewis antigen decreased at later stages, which was validated by Aleuria aurantia lectin (AAL) and Ulex europaeus lectin (UEA-I) blots. We also used HPLC coupled with tandem mass spectrometry to identify 611 N-glycosylation sites on 350 N-glycoproteins at the early stage developmental stage 1 (fertilized egg), and 1682 N-glycosylation sites on 1023 N-glycoproteins at stage 41 (late tailbud stage). Over two thirds of the N-glycoproteins identified in the late tailbud stage are associated with neuron projection morphogenesis, suggesting a vital role of the N-glycome in neuronal development.

A surface-enhanced Raman spectroscopy database of 63 metabolites.

Metabolomics, the study of metabolic profiles in a biological sample, has seen rapid growth due to advances in measurement technologies such as mass spectrometry (MS). While MS metabolite reference libraries have been generated for metabolomics applications, mass spectra alone are unable to unambiguously identify many metabolites in a sample; these unidentified compounds are typically annotated as "features". Surface-enhanced Raman spectroscopy (SERS) is an interesting technology for metabolite identification based on vibrational spectra. However, no reports have been published that present SERS metabolite spectra from chemical libraries. In this paper, we demonstrate that an untargeted approach utilizing citrate-capped silver nanoparticles yields SERS spectra for 20% of 80 compounds chosen randomly from a commercial metabolite library. Furthermore, prescreening of the metabolites according to chemical functionality allowed for the efficient identification of samples within the library that yield distinctive SERS spectra under our experimental conditions. Last, we present a reference database of 63 metabolite SERS spectra for use as an identification tool in metabolomics studies; this set includes 30 metabolites that have not had previously published SERS spectra.

Database of free solution mobilities for 276 metabolites.

Although databases are available that provide mass spectra and chromatographic retention information for small-molecule metabolites, no publicly available database provides electrophoretic mobility for common metabolites. As a result, most compounds found in electrophoretic-based metabolic studies are unidentified and simply annotated as "features". To begin to address this issue, we analyzed 460 metabolites from a commercial library using capillary zone electrophoresis coupled with electrospray mass spectrometry. To speed analysis, a sequential injection method was used wherein six compounds were analyzed per run. An uncoated fused silica capillary was used for the analysis at 20 °C with a 0.5% (v/v) formic acid and 5% (v/v) methanol background electrolyte. A Prince autosampler was used for sample injection and the capillary was coupled to an ion trap mass spectrometer using an electrokinetically-pumped nanospray interface. We generated mobility values for 276 metabolites from the library (60% success rate) with an average standard deviation of 0.01 × 10-8 m2V-1s-1. As expected, cationic and anionic compounds were well resolved from neutral compounds. Neutral compounds co-migrated with electro-osmotic flow. Most of the compounds that were not detected were neutral and presumably suffered from adsorption to the capillary wall or poor ionization efficiency.

MALDI-imaging of early stage Xenopus laevis embryos.

Xenopus laevis is an important model organism for vertebrate development. An extensive literature has developed on changes in transcript expression during development of this organism, and there is a growing literature on the corresponding protein expression changes during development. In contrast, there is very little information on changes in metabolite expression during development. We present the first MALDI mass-spectrometry images of metabolites within the developing embryo. These images were generated for 142 metabolite ions. The images were subjected to an algorithm that revealed three spatially-resolved clusters of metabolites. One small cluster is localized near the outer membrane of the embryo. A large cluster of metabolites is found in cavities destined to form the neural tube and gut, and contains a number of ceramide species, which are associated with cellular signaling, including differentiation, proliferation, and programmed cell death. Another large cluster of metabolites is found in tissue and is dominated by phosphatidylcholines, which are common components of cell membranes. Surprisingly, no metabolites appear to be homogeneously distributed across the slices; metabolites are localized either within tissue or in cavities, but not both.

Detection of 1 zmol injection of angiotensin using capillary zone electrophoresis coupled to a Q-Exactive HF mass spectrometer with an electrokinetically pumped sheath-flow electrospray interface.

An electrokinetically pumped sheath-flow nanoelectrospray interface provides an efficient means of transferring ions from a capillary electrophoresis system into a mass spectrometer. To characterize its performance, we analyzed angiotensin solutions prepared in a background of 0.25 mg/mL of a BSA tryptic digest. Calibration curves were prepared from 10 zmol (10-21 mol) to 10 fmol (10-14 mol) of angiotensin injected into the capillary. A parallel reaction monitoring approach was used; MS1 was set to m/z = 523.8 ±â€¯2, and fragment ion signals at 263.1389 (y2+) and 784.4095 (b6+) were used to generate selected ion electropherograms. Calibration curves based on peak areas were linear (log-log slope of 0.94 for the y2+ fragment and 0.98 for the b6+ fragment). We then injected 1-zmol (600 copies) of angiotensin in the BSA background using a 10-µm ID separation capillary. Triplicate analyses consistently produced co-migrating peaks for the two fragment ions. The b6+ electropherogram showed no background signal, whereas the y2+ electropherogram showed a few noise spikes that were smaller than the peak maximum. Bienayme-Tchebycheff inequality was used to estimate detection limits of 230  ymol (140 ions) injected into the separation capillary. To the best of our knowledge, these electropherograms present the smallest number of molecules detected using mass spectrometry coupled with a separation.

Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry Produces over 4400 Phosphopeptide Identifications from a 220 ng Sample.

The dependence of capillary zone electrophoresis (CZE) separations on the charge state of the analyte is useful for the analysis of many post-translational modifications in proteins. In this work, we coupled CZE to an Orbitrap Fusion Lumos Tribrid platform with an advanced peak determination algorithm for phosphoproteomics analysis. A linear-polyacrylamide-coated capillary with very low electroosmotic flow was used for the separation. The optimal injection volume was between 100 and 150 nL of a solution of phosphopeptides in 30 mM ammonium bicarbonate (pH 8.2) buffer, which produces a dynamic pH junction sample injection. Larger injection volumes resulted in serious peak broadening and decreased numbers of phosphopeptide identifications. The optimized system identified 4405 phosphopeptides from 220 ng of enriched phosphopeptides from mouse brain, which represents the state-of-the-art result for single-shot CZE-ESI-MS/MS-based phosphoproteome analysis. We found that the migration time for phosphopeptides is much longer than that for non-phosphopeptides and increased along with the number of phosphorylation sites on the peptides, as expected for the additional negative charges associated with the phosphate groups. We also investigated the phosphorylation site motifs; a number of motifs appeared in the CZE-ESI-MS/MS data but not in LC-ESI-MS/MS data, which suggested the complementary performance of the techniques. The data are available via ProteomeXchange with identifier PXD012888.

Capillary Zone Electrophoresis with Fraction Collection for Separation, Culturing, and Identification of Bacteria from an Environmental Microbiome.

Capillary zone electrophoresis (CZE) can produce high-resolution separations of biological samples, including microbial mixtures. The study of complex populations of microorganisms using CZE is limited because most detectors have limited sensitivity, are destructive, and provide limited information for microbial identification. To address these issues, we developed an integrated capillary zone electrophoresis apparatus to fractionate bacteria from complex mixtures. We deposited fractions onto nutrient agar in a Petri dish for microbial culturing, and we subjected the observed colonies to Sanger sequencing of a phylogenetic marker, the 16S rRNA gene, for microbial identification. We separated and cultured both a single bacteria species, the model Gram-negative organism Escherichia coli, and a complex environmental isolate of primary sewage effluent. Sequence analysis of the 16S rRNA genes from this mixture identified 15 ± 5 distinct bacterial species per run. This approach requires minimal manipulation of microbial populations and combines electrophoretic fractionation of bacterial cells with automated collection for accurate identification of species. This approach should be applicable to microorganisms in general and may enable discrimination of physiologically different cells in complex assemblages, such as in microbiome samples.

Author Correction: Phosphorylation Dynamics Dominate the Regulated Proteome during Early Xenopus Development.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Production of Over 27 000 Peptide and Nearly 4400 Protein Identifications by Single-Shot Capillary-Zone Electrophoresis-Mass Spectrometry via Combination of a Very-Low-Electroosmosis Coated Capillary, a Third-Generation Electrokinetically-Pumped Sheath-Flow Nanospray Interface, an Orbitrap Fusion Lumos Tribrid Mass Spectrometer, and an Advanced-Peak-Determination Algorithm.

We show that capillary-zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) generates very large numbers of peptide and protein identifications (IDs) by combining four technologies: a separation capillary coated to generate very low electroosmosis, an electrokinetically pumped sheath-flow nanoelectrospray interface to produce high-sensitivity ionization, an Orbitrap Fusion Lumos Tribrid platform to provide high-speed analysis, and an advanced-peak-determination (APD) algorithm to take advantage of the mass spectrometer's data-acquisition speed. The use of the APD algorithm resulted in 2 times more identifications than the standard peak algorithm. We also investigated the effect of the isolation window, injection time, and loading amount. Optimization of these parameters produced over 27 000 peptide identifications and nearly 4400 protein-group identifications from 220 ng of K562-cell digest in a single 120 min run, which is 2.7 times more IDs produced by CZE-ESI-MS/MS than by the previous state-of-the-art technique.

Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli.

For many proteins, aggregation is one part of a structural equilibrium that can occur. Balancing productive aggregation versus pathogenic aggregation that leads to toxicity is critical and known to involve adenosine triphosphate (ATP) dependent action of chaperones and disaggregases. Recently a second activity of ATP was identified, that of a hydrotrope which, independent of hydrolysis, was sufficient to solubilize aggregated proteins in vitro. This novel function of ATP was postulated to help regulate proteostasis in vivo. We tested this hypothesis on aggregates found in Xenopus oocyte nucleoli. Our results indicate that ATP has dual roles in the maintenance of protein solubility. We provide evidence of endogenous hydrotropic action of ATP but show that hydrotropic solubilization of nucleolar aggregates is preceded by a destabilizing event. Destabilization is accomplished through an energy dependent process, reliant upon ATP and one or more soluble nuclear factors, or by disruption of a co-aggregate like RNA.

Metabolomics of oncogene-specific metabolic reprogramming during breast cancer.

BACKGROUND: The complex yet interrelated connections between cancer metabolism and oncogenic driver genes are relatively unexplored but have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. The goal of this study was to identify global metabolic profiles of breast tumors isolated from multiple transgenic mouse models and to identify unique metabolic signatures driven by these oncogenes. METHODS: Using mass spectrometry (GC-MS, LC-MS/MS, and capillary zone electrophoresis (CZE)-MS platforms), we quantified and compared the levels of 374 metabolites in breast tissue from normal and transgenic mouse breast cancer models overexpressing a panel of oncogenes (PyMT, PyMT-DB, Wnt1, Neu, and C3-TAg). We also compared the mouse metabolomics data to published human metabolomics data already linked to clinical data. RESULTS: Through analysis of our metabolomics data, we identified metabolic differences between normal and tumor breast tissues as well as metabolic differences unique to each initiating oncogene. We also quantified the metabolic profiles of the mammary fat pad versus mammary epithelium by CZE-MS/MS. However, the differences between the tissues did not account for the majority of the metabolic differences between the normal mammary gland and breast tumor tissues. Therefore, the differences between the cohorts were unlikely due to cellular heterogeneity. Of the mouse models used in this study, C3-TAg was the only cohort with a tumor metabolic signature composed of ten metabolites that had significant prognostic value in breast cancer patients. Gene expression analysis identified candidate genes that may contribute to the metabolic reprogramming. CONCLUSIONS: This study identifies oncogene-induced metabolic reprogramming within mouse breast tumors and compares the results to that of human breast tumors, providing a unique look at the relationship between and clinical value of oncogene initiation and metabolism during breast cancer.