Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling

The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., approximately 10 000 cells, which typically contain roughly 1-4 microg protein. The 16O/18O labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and 16O/18O labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the 16O/18O post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 microg. Next, protein expression was compared between 10 000 cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then 16O/18O labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate dehydrogenase, actin and 14-3-3 protein xi/delta were found to be significantly up-regulated in the breast tumor cells.     

Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot

Potato is the world's largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora. Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.     

Cutting Edge: Multiple autoimmune pathways in kd/kd mice

The kidney disease (kd) mutation was transferred to a C57BL/6 (B6) background by selection for closely linked microsatellite markers. The resulting congenic strain, B6.kd, was mated with partners homozygous for targeted mutations of CD4, CD8, CD28, IL-2, recombinase-activating gene-1 (Rag-1), ICAM-1, or beta(2)-microglobulin. In most of the resulting double mutants, kidney disease occurred as readily and as severely as in the B6.kd controls, although disease occurred somewhat less frequently in age-matched CD28(-/-) kd/kd mice. Immunohistology demonstrated a predominance of macrophages in the lesions of B6.kd and most of the double mutants, with the remaining cells consisting of T cells and variable numbers of NK cells. In Rag-1(-/-) kd/kd, approximately 50% of infiltrating cells were macrophages, and approximately 50% were NK cells. These results suggest that the initial lesion caused by the mutant gene is intrinsic to the kidney and that the immune response that subsequently occurs can involve any one of several different cellular compositions.     

Structure-activity relationships in the hydrophobic interactions of polyphenols with cellulose and collagen

Polyphenol interactions with both cellulose and collagen in the solid state have been studied by using chromatography on cellulose and by evaluating the hydrothermal stability of the polyphenol treated sheepskin collagen. Twenty-four polyphenolic compounds were studied, including seven glucose-based gallotannins, five polyalcohol-based gallotannins, and twelve ellagitannins. In the cellulose-polyphenols systems, the polyphenol's affinity to cellulose is positively correlated with their molecular masses, the number of galloyl groups, and their hydrophobicity (logP). The polyphenol treatment increased the hydrothermal stability of collagen samples, and such effects are also positively correlated with the molecular masses, total number of galloyl groups and the hydrophobicity of polyphenols. Ellagitannins showed much weaker interactions with both biopolymers than gallotannins having similar molecular mass, the same number of galloyl groups, and the same number of phenolic hydroxyl groups. It is concluded that, for the polyphenol interactions with both cellulose and collagen, (1) the galloyl group of polyphenols is the functional group; (2) the strength of interactions are positively correlated with molecular size, the number of galloyl groups and the hydrophobicity of polyphenols; (3) the hydrophobic interactions are of great significance; and (4) the interactions are strongly dependent on the flexibility of galloyl groups.     

Androgen receptor phosphorylation. Regulation and identification of the phosphorylation sites

Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of cross-talk between growth factor signaling and androgen in prostate development, physiology, and cancer.     

Chlorxanthomycin,a fluorescent,chlorinated, pentacyclic pyrene from a Bacillus sp

A gram-positive Bacillus sp. that fluoresces yellow under long-wavelength UV light on several common culture media was isolated from soil samples. On the basis of carbon source utilization studies, fatty acid methyl ester analysis, and 16S ribosomal DNA analysis, this bacterium was most similar to Bacillus megaterium. Chemical extraction yielded a yellow-orange fluorescent pigment, which was characterized by X-ray crystallography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The fluorescent compound, chlorxanthomycin, is a pentacyclic, chlorinated molecule with the molecular formula C22H15O6Cl and a molecular weight of 409.7865. Chlorxanthomycin appears to be located in the cytoplasm, does not diffuse out of the cells into the culture medium, and has selective antibiotic activity.     

Hydrogen peroxide and nitric oxide as signalling molecules in plants

It is now clear that hydrogen peroxide (H(2)O(2)) and nitric oxide (NO) function as signalling molecules in plants. A wide range of abiotic and biotic stresses results in H(2)O(2) generation, from a variety of sources. H(2)O(2) is removed from cells via a number of antioxidant mechanisms, both enzymatic and non-enzymatic. Both biotic and abiotic stresses can induce NO synthesis, but the biosynthetic origins of NO in plants have not yet been resolved. Cellular responses to H(2)O(2) and NO are complex, with considerable cross-talk between responses to several stimuli. In this review the potential roles of H(2)O(2) and NO during various stresses and the signalling pathways they activate are discussed. Key signalling components that might provide targets for enhancing crop production are also identified.     

Targeted proteomics of low-level proteins in human plasma by LC/MSn: using human growth hormone as a model system

This paper describes the profiling of human growth hormone (hGH) in human plasma in order to assess the dynamic range of the ion-trap mass spectrometer for proteomic studies of complex biological samples. Human growth hormone is an example of a low-level plasma protein in vivo, present at subfemtomole levels. This study was performed on a plasma sample in which hGH has been spiked at 10-fold above the natural level, that is approximately 16 pg/microL of plasma. Initially, the measurement was carried out without any sample enrichment and consisted of the following steps: the full set of plasma proteins were reduced, alkylated, and digested with trypsin, and the resulting peptides were separated on a capillary C-18 column and then detected by ion-trap mass spectrometry (1D LC/MS). In addition, this study provided a global view of the serum proteome with over 200 plasma proteins being preliminarily identified. In the MS/MS analysis, hGH was detected by characterization of the first tryptic peptide (T1). The initial identification was confirmed by alternative approaches, which also allowed the evaluation of different sample purification protocols. First, the plasma sample containing hGH was fractionated on a reversed-phase HPLC column and digested, and hGH could now be identified by MS/MS measurements of two tryptic peptides (T1 and T4) by the same 1D LC/MS protocol. In addition, the assignment of peptide identity was made with higher certainty (as measured by an algorithm score). The plasma sample was also fractionated by 1D and 2D gel electrophoresis, the selected bands were digested and analyzed again by the 1D LC/MS protocol. In both cases using the gel prepurifications, hGH was identified with additional peptides. Finally, the plasma sample was analyzed by 2D chromatography (ion exchange and reversed phase) on a new instrumental platform (ProteomeX), and hGH was identified by the observation of five tryptic peptides. In conclusion, these experiments were able to detect growth hormone in the low femtomole level with a dynamic range of 1 in 40 000 by several independent approaches. The amount of growth hormone, while 10-fold above normal in vivo levels, represents concentrations that may be present in disease states (such as acromegaly) and also in doping control measurements. These studies have demonstrated that shotgun sequencing approaches (LC/MS/MS) not only can profile high-abundance proteins in complex biological fluids but also have the potential to identify and quantitate low-level proteins present in such complex mixtures without extensive prepurification protocols. A key to such studies, however, is to use targeted approaches that reduce the complexity of the solute mixture that is presented to the mass spectrometer at a given time point. The various sample preparation protocols described here all improved the quality of the hGH measurement, although in this study the 2D chromatographic approach gave the greatest sequence coverage.