Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

Generation of red fluorescent protein transgenic dogs

Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene‐expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP‐fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. genesis 47:314–322, 2009. © 2009 Wiley‐Liss, Inc.     

Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteins

Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology.     

Isolation and characterization of 17 microsatellite loci for the house finch (Carpodacus mexicanus)

The house finch (Carpodacus mexicanus) has emerged recently as a model species in studies of sexual selection, reproductive physiology, population genetics, and epizootic disease ecology. Here we describe 17 highly polymorphic microsatellite loci for this species. In a sample of 36 individuals, we observed an average of 16 alleles per locus and heterozygosity ranged from 0.61 to 0.97. One locus showed significant deviation from Hardy-Weinberg proportions, but no significant gametic disequilibrium was observed among any of the loci. Amplification by polymerase chain reaction was optimized under similar parameters across loci, thereby facilitating multiplexing and rapid multilocus genotyping.     

Long-term microcarrier suspension cultures of human embryonic stem cells

The conventional method of culturing human embryonic stem cells (hESC) is on two-dimensional (2D) surfaces, which is not amenable for scale up to therapeutic quantities in bioreactors. We have developed a facile and robust method for maintaining undifferentiated hESC in three-dimensional (3D) suspension cultures on matrigel-coated microcarriers achieving 2- to 4-fold higher cell densities than those in 2D colony cultures. Stable, continuous propagation of two hESC lines on microcarriers has been demonstrated in conditioned media for 6 months. Microcarrier cultures (MC) were also demonstrated in two serum-free defined media (StemPro and mTeSR1). MC achieved even higher cell concentrations in suspension spinner flasks, thus opening the prospect of propagation in controlled bioreactors.     

Enhancing Identifications of Lipid-embedded Proteins by Mass Spectrometry for Improved Mapping of Endothelial Plasma Membranes in Vivo

Lipid membranes structurally define the outer surface and internal organelles of cells. The multitude of proteins embedded in lipid bilayers are clearly functionally important, yet they remain poorly defined. Even today, integral membrane proteins represent a special challenge for current large scale shotgun proteomics methods. Here we used endothelial cell plasma membranes isolated directly from lung tissue to test the effectiveness of four different mass spectrometry-based methods, each with multiple replicate measurements, to identify membrane proteins. In doing so, we substantially expanded this membranome to 1,833 proteins, including >500 lipid-embedded proteins. The best method combined SDS-PAGE prefractionation with trypsin digestion of gel slices to generate peptides for seamless and continuous two-dimensional LC/MS/MS analysis. This three-dimensional separation method outperformed current widely used two-dimensional methods by significantly enhancing protein identifications including single and multiple pass transmembrane proteins; >30% are lipid-embedded proteins. It also profoundly improved protein coverage, sensitivity, and dynamic range of detection and substantially reduced the amount of sample and the number of replicate mass spectrometry measurements required to achieve 95% analytical completeness. Such expansion in comprehensiveness requires a trade-off in heavy instrument time but bodes well for future advancements in truly defining the ever important membranome with its potential in network-based systems analysis and the discovery of disease biomarkers and therapeutic targets. This analytical strategy can be applied to other subcellular fractions and should extend the comprehensiveness of many future organellar proteomics pursuits.The plasma membrane provides a fundamental physical interface between the inside and outside of any cell. Beyond creating a protected compartment with a segregated, distinct, and well controlled internal milieu for the cell, it also mediates a wide variety of basic biological functions including signal transduction, molecular transport, membrane trafficking, cell migration, cell-cell interactions, intercellular communication, and even drug resistance. Plasma membrane-associated proteins, especially integral membrane proteins (IMPs)¹ that traverse the lipid bilayer, are key elements mediating these vital biological processes. Consistent with its fundamental importance in both normal cellular functions and pathophysiology, the plasma membrane has also been targeted extensively for biomarker discovery and drug development. In fact, more than two-thirds of known targets for existing drugs are plasma membrane proteins (1).Despite the potential benefits, profiling the proteome of plasma membranes comprehensively using standard large scale methods including MS-based strategies has been limited and technically quite challenging. Intrinsic hydrophobicity, a wide concentration range of proteins, and other factors have hampered IMP resolution and identification using conventional two-dimensional gel electrophoresis. Gel and gel-free protein separations, including combinations of both, have been reported as an alternative to two-dimensional gel electrophoresis (2–9). Yet most such efforts have focused predominantly on identifying rather soluble proteins from body fluids (i.e. plasma, serum, and cerebrospinal fluid), cell lysates, or cytoplasm. These proteins, unlike IMPs, are relatively abundant and readily susceptible to enzymatic digestion in solution. Various attempts have been made to solubilize and enrich for IMPs, including different detergents, solvents, high pH solutions, and affinity purification (10–22). Even when organellar membranes are enriched through isolation by subcellular fractionation, the yield of proteins identified has been below expectation, especially for multipass transmembrane proteins such as G-protein-coupled receptors.Here we systematically characterize four analytical approaches to enhance the identification of proteins, specifically those embedded in plasma membranes isolated directly from vascular endothelium in rat lung. Endothelial cells (ECs) constitute the tissue-blood interface that controls many important physiological functions, including tissue homeostasis, nutrition, vasomotion, and even drug delivery. In vivo mapping of the EC plasma membrane proteome provides unique opportunities for extending basic understanding in vascular biology and for directing the delivery of therapeutic and imaging agents in vivo (23–25). But it also presents distinct challenges beyond those generally associated with extraction, solubilization, and identification of IMPs in cells and tissues. ECs form a thin monolayer lining each blood vessel. They constitute a very small fraction of all the cells existing in tissue, thereby making it difficult to isolate sufficiently pure EC plasma membrane fractions for proteomics analysis using conventional subcellular fractionation techniques. Although relatively simple to isolate from tissue and grow in culture, ECs require cues from the tissue microenvironment to maintain their tissue-specific qualities and thus undergo rapid and considerable phenotypic drift after isolation (26).We have developed a specialized coating procedure using colloidal silica nanoparticles perfused through the blood vessels of the tissue to isolate luminal plasma membranes of the vascular endothelium as they exist natively in tissue (26–28). Our initial survey of these plasma membranes isolated directly from rat lungs used primarily three standard analytical techniques of the time: two-dimensional electrophoresis, Western analysis, and the shotgun method of two-dimensional liquid chromatography-tandem mass spectrometry (24, 26). We identified 450 proteins of which only ∼15% were IMPs. Although at the time this was a notable total number of proteins, more IMPs are expected. In fact, this large scale 2DC study did not identify several well known EC surface marker proteins, including specific enzymes, adhesion molecules, and growth factor receptors.Here we comparatively analyze four different MS-based strategies involving two- and three-dimensional separation by combining protein prefractionation via SDS-PAGE with in-gel digestion to produce peptides separated by one- and two-dimensional nano-HPLC before seamless and continuous MS analysis. Each method used multiple replicate measurements to comprehensively identify proteins, especially IMPs, and in doing so achieved a clear statistical definition of completeness that permits meaningful comparisons. Ultimately this analysis greatly expanded the EC plasma membranome to 1,833 proteins of which nearly 30% are membrane-embedded.     

Preventive effect of piperonyl butoxide on cyclophosphamide-induced teratogenesis in rats

BACKGROUND: Cyclophosphamide induces fetal defects through metabolic activation by cytochrome P-450 monooxygenases (CYP). The effects of piperonyl butoxide (PBO), a CYP inhibitor, on the fetal development and external, visceral, and skeletal abnormalities induced by cyclophosphamide were investigated in rats. METHODS: Pregnant rats were daily administered PBO (400 mg/kg) by gavage for 7 days (the 6th to 12th day of gestation), and intraperitoneally administered with cyclophosphamide (12 mg/kg) 4 h after the final treatment. On the 20th day of gestation, maternal and fetal abnormalities were determined by Cesarean section. RESULTS: Cyclophosphamide reduced fetal body weights by 30–40% without increasing resorption or death. In addition, it induced malformations in live fetuses: 100, 98, and 98.2% of the external (head and limb defects), visceral (cerebroventricular dilatation, cleft palate, and renal pelvic/ureteric dilatation), and skeletal (acrania, vertebral/costal malformations, and delayed ossification) abnormalities, respectively. The pre-treatment of PBO greatly decreased mRNA expression and activity of hepatic CYP2B, which metabolizes cyclophosphamide into teratogenic acrolein and cytotoxic phosphoramide mustard. Moreover, PBO remarkably attenuated cyclophosphamide-induced body weight loss and abnormalities of fetuses; score 3.57 versus 1.87 for exencephaly, 75.5% versus 42.5% for limb defects, 65.3% versus 22% for cerebroventricular dilatation, 59.2% versus 5.1% for cleft palate, score 1.28 versus 0.93 for renal pelvic/ureteric dilatation, 71.9–82.5% versus 23–45.9% for vertebral/costal malformations, and 84.2% versus 57.4% for delayed ossification in cyclophosphamide alone and PBO co-administration groups. CONCLUSIONS: These results suggest that repeated treatment with PBO may improve cyclophosphamide-induced body weight loss and malformations of fetuses by down-regulating CYP2B. Birth Defects Res (Part B) 86:402–408, 2009. © 2009 Wiley-Liss, Inc.