Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA) and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT) method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species

pH-Responsive Artemisinin Derivatives and Lipid Nanoparticle Formulations Inhibit Growth of Breast Cancer Cells <em>In Vitro</em> and Induce Down-Regulation of HER Family Members

<div><p>Artemisinin (ART) dimers show potent anti-proliferative activities against breast cancer cells. To facilitate their clinical development, novel pH-responsive artemisinin dimers were synthesized for liposomal nanoparticle formulations. A new ART dimer was designed to become increasingly water-soluble as pH declines. The new artemisinin dimer piperazine derivatives (ADPs) remained tightly associated with liposomal nanoparticles (NPs) at neutral pH but were efficiently released at acidic pH's that are known to exist within solid tumors and organelles such as endosomes and lysosomes. ADPs incorporated into nanoparticles down regulated the anti-apoptotic protein, survivin, and cyclin D1 when incubated at low concentrations with breast cancer cell lines. We demonstrate for the first time, for any ART derivative, that ADP NPs can down regulate the oncogenic protein HER2, and its counterpart, HER3 in a HER2+ cell line. We also show that the wild type epidermal growth factor receptor (EGFR or HER1) declines in a triple negative breast cancer (TNBC) cell line in response to ADP NPs. The declines in these proteins are achieved at concentrations of NP109 at or below 1 µM. Furthermore, the new artemisinin derivatives showed improved cell-proliferation inhibition effects compared to known dimer derivatives.</p> </div

Summary of IC₅₀ values calculated from MTT assays of ADPs and NPs on BT474 and MDA-MB-231 cells.

<p>Values represent average (±SD) calculated from three independent experiments. *Exceeded maximum concentration of assay.</p

Summary of loading and release efficiencies of the NPs.

<p>Values represent an average and standard deviation of three independent experiments read at λ = 263 nm.</p

Effects of NP109 on the expression of selected proteins involved in cell proliferation, cell cycling, and apoptosis in BT474 (a–c) and MDA-MB-231 cells (d–f).

<p>Effects of NP109 on the expression of selected proteins involved in cell proliferation, cell cycling, and apoptosis in BT474 (a–c) and MDA-MB-231 cells (d–f).</p