Person:

Breitkopf, Susanne B.

Using a series of immunoprecipitation (IP) – tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

This study demonstrates a liquid-liquid extraction for the sequential tandem mass spectrometry (LC-MS/MS) analysis of non-polar lipids, polar metabolites, proteins and phosphorylation sites from a single piece of tissue. Extraction of 10 mg BRCA−/−, p53−/− breast tumor tissue or normal mammary gland tissue with methyl-tert-butyl ether (MTBE) results in three phases: an upper non-polar phase containing 1,382 lipids, a lower polar phase with 805 metabolites and a precipitated protein pellet with 4,792 proteins with 1,072 phosphorylation sites. Comparative analysis revealed an activated AKT-mTOR pathway in tumors. Tumors also showed a reduction of phosphorylation sites involved in transcription and RNA splicing and decreased abundance of enzymes in lipid synthesis. Analysis of polar metabolites revealed a reduction in glycolysis, pentose phosphate pathway, polyamines and nucleotides, but an increase in TCA and urea cycle intermediates. Analysis of lipids revealed a shift from high triglycerides in mammary gland to high phospholipid levels in tumors. The data were integrated into a model showing breast tumors exhibit features on the proteomic, lipidomic and metabolomic level that are distinct from normal breast tissue. Our integrative technique lends itself to samples such as tumor biopsies, dried blood spots and fluids including urine and CSF to develop biomarkers of disease.

Horse urine is easily collected and contains molecules readily measurable using mass spectrometry that can be used as biomarkers representative of health, disease or drug tampering. This study aimed at analyzing microliter levels of horse urine to purify, identify and quantify proteins, polar metabolites and non-polar lipids. Urine from a healthy 12 year old quarter horse mare on a diet of grass hay and vitamin/mineral supplements with limited pasture access was collected for serial-omics characterization. The urine was treated with methyl tert-butyl ether (MTBE) and methanol to partition into three distinct layers for protein, non-polar lipid and polar metabolite content from a single liquid-liquid extraction and was repeated two times. Each layer was analyzed by high performance liquid chromatography—high resolution tandem mass spectrometry (LC-MS/MS) to obtain protein sequence and relative protein levels as well as identify and quantify small polar metabolites and lipids. The results show 46 urine proteins, many related to normal kidney function, structural and circulatory proteins as well as 474 small polar metabolites but only 10 lipid molecules. Metabolites were mostly related to urea cycle and ammonia recycling as well as amino acid related pathways, plant diet specific molecules, etc. The few lipids represented triglycerides and phospholipids. These data show a complete mass spectrometry based—omics characterization of equine urine from a single 333 μL mid-stream urine aliquot. These omics data help serve as a baseline for healthy mare urine composition and the analyses can be used to monitor disease progression, health status, monitor drug use, etc.