Timothy J. Barder

Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates.

A review of two-dimensional (2D) liquid separation methods used in our laboratory to map the protein content of human cancer cells is presented herein. The methods discussed include various means of fractionating proteins according to isoelectric point (pI) in the first dimension. The proteins in each pI fraction are subsequently separated using nonporous (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC). The liquid eluent of the RP-HPLC separation is directed on-line into an electrospray ionization time-of-flight (ESI-TOF) mass spectrometer where an accurate value of the protein intact M(r) can be obtained. The result is a 2D map of pI versus M(r) analogous to 2D gel electrophoresis; however the highly accurate and reproducible M(r) serves as the basis for interlysate comparisons. In addition, the use of liquid separations allows for the collection of hundreds of purified proteins in the liquid phase for further analysis via peptide mass mapping using matrix assisted laser desorption ionization TOF MS. A description of the methodology used and its applications to analysis of several types of human cancer cell lines is described. The potential of the method for differential proteomic analysis for the identification of biomarkers of disease is discussed.

Insight into PreImplantation Factor (PIF*) Mechanism for Embryo Protection and Development: Target Oxidative Stress and Protein Misfolding (PDI and HSP) through Essential RIPK Binding Site

Background Endogenous PIF, upon which embryo development is dependent, is secreted only by viable mammalian embryos, and absent in non-viable ones. Synthetic PIF (sPIF) administration promotes singly cultured embryos development and protects against their demise caused by embryo-toxic serum. To identify and characterize critical sPIF-embryo protein interactions novel biochemical and bio-analytical methods were specifically devised. Methods FITC-PIF uptake/binding by cultured murine and equine embryos was examined and compared with scrambled FITC-PIF (control). Murine embryo (d10) lysates were fractionated by reversed-phase HPLC, fractions printed onto microarray slides and probed with Biotin-PIF, IDE and Kv1.3 antibodies, using fluorescence detection. sPIF-based affinity column was developed to extract and identify PIF-protein interactions from lysates using peptide mass spectrometry (LC/MS/MS). In silico evaluation examined binding of PIF to critical targets, using mutation analysis. Results PIF directly targets viable cultured embryos as compared with control peptide, which failed to bind. Multistep Biotin-PIF targets were confirmed by single-step PIF-affinity column based isolation. PIF binds protein disulfide isomerases a prolyl-4-hydroxylase β-subunit, (PDI, PDIA4, PDIA6-like) containing the antioxidant thioredoxin domain. PIF also binds protective heat shock proteins (70&90), co-chaperone, BAG-3. Remarkably, PIF targets a common RIPK site in PDI and HSP proteins. Further, single PIF amino acid mutation significantly reduced peptide-protein target bonding. PIF binds promiscuous tubulins, neuron backbones and ACTA-1,2 visceral proteins. Significant anti-IDE, while limited anti-Kv1.3b antibody-binding to Biotin-PIF positive lysates HPLC fractions were documented. Conclusion Collectively, data identifies PIF shared targets on PDI and HSP in the embryo. Such are known to play a critical role in protecting against oxidative stress and protein misfolding. PIF-affinity-column is a novel utilitarian method for small molecule targets direct identification. Data reveals and completes the understanding of mechanisms involved in PIF-induced autotrophic and protective effects on the embryo.

Classification of Cancer Cell Lines Using an Automated Two-dimensional Liquid Mapping Method with Hierarchical Clustering Techniques

A two-dimensional liquid mapping method was used to map the protein expression of eight ovarian serous carcinoma cell lines and three immortalized ovarian surface epithelial cell lines. Maps were produced using pI as the separation parameter in the first dimension and hydrophobicity based upon reversed-phase HPLC separation in the second dimension. The method can be reproducibly used to produce protein expression maps over a pH range from 4.0 to 8.5. A dynamic programming method was used to correct for minor shifts in peaks during the HPLC gradient between sample runs. The resulting corrected maps can then be compared using hierarchical clustering to produce dendrograms indicating the relationship between different cell lines. It was found that several of the ovarian surface epithelial cell lines clustered together, whereas specific groups of serous carcinoma cell lines clustered with each other. Although there is limited information on the current biology of these cell lines, it was shown that the protein expression of certain cell lines is closely related to each other. Other cell lines, including one ovarian clear cell carcinoma cell line, two endometrioid carcinoma cell lines, and three breast epithelial cell lines, were also mapped for comparison to show that their protein profiles cluster differently than the serous samples and to study how they cluster relative to each other. In addition, comparisons can be made between proteins differentially expressed between cell lines that may serve as markers of ovarian serous carcinomas. The automation of the method allows reproducible comparison of many samples, and the use of differential analysis limits the number of proteins that might require further analysis by mass spectrometry techniques.

PreImplantation factor (PIF*) regulates systemic immunity and targets protective regulatory and cytoskeleton proteins.

Secreted by viable embryos, PIF is expressed by the placenta and found in maternal circulation. It promotes implantation and trophoblast invasion, achieving systemic immune homeostasis. Synthetic PIF successfully transposes endogenous PIF features to non-pregnant immune and transplant models. PIF affects innate and activated PBMC cytokines and genes expression. We report that PIF targets similar proteins in CD14+, CD4+ and CD8+ cells instigating integrated immune regulation. PIF-affinity chromatography followed by mass-spectrometry, pathway and heatmap analysis reveals that SET-apoptosis inhibitor, vimentin, myosin-9 and calmodulin are pivotal for immune regulation. PIF acts on macrophages down-stream of LPS (lipopolysaccharide-bacterial antigen) CD14/TLR4/MD2 complex, targeting myosin-9, thymosin-α1 and 14-3-3eta. PIF mainly targets platelet aggregation in CD4+, and skeletal proteins in CD8+ cells. Pathway analysis demonstrates that PIF targets and regulates SET, tubulin, actin-b, and S100 genes expression. PIF targets systemic immunity and has a short circulating half-life. Collectively, PIF targets identified; protective, immune regulatory and cytoskeleton proteins reveal mechanisms involved in the observed efficacy against immune disorders.

Proteomic Analysis Using 2‐D Liquid Separations of Intact Proteins From Whole‐Cell Lysates

This unit describes procedures for 2‐D liquid separations of proteins from whole‐cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI‐based fractionation using a batch‐phase electrophoretic method, while chromatofocusing uses a column‐based chromatographic method to generate the pH gradient. Using either method, a second‐dimension fractionation is provided in the liquid phase using nonporous silica‐based reversed‐phase HPLC (NPS‐RP‐HPLC) to generate a 2‐D liquid map of the protein content of the cell. The eluate of the 2‐D liquid fractionation is directly coupled to a mass spectrometer for on‐line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.