Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry.

Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1-5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.

Altered protein expression at early-stage rat hepatic neoplasia.

Protein expression patterns were analyzed in a rat model of hepatic neoplasia to detect changes reflecting biological mechanism or potential therapeutic targets. The rat resistant hepatocyte model of carcinogenesis was studied, with a focus on the earliest preneoplastic lesion visible in the liver, the preneoplastic hyperplastic nodule. Expression differences were shown by two-dimensional polyacrylamide gel electrophoresis and image analysis. Polypeptide masses were measured by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) and their sequences were obtained by tandem mass spectrometry. Alterations in expression of cytoskeletal and functional proteins were demonstrated, consistent with biological changes known to occur in the preneoplastic cells. Of particular interest was the differential expression of a serine protease inhibitor (serpin) with a role implicated in angiogenesis. Serpin, implicated in the inhibition of angiogenesis, is present in normal liver but has greatly reduced expression at the preneoplastic stage of liver cancer development. Immunofluorescence microscopy with antibodies to this serpin, kallistatin, supports the proteomic identification. Immunofluorescence microscopy with antibodies to the blood vessel marker von Willebrand factor provides evidence for neovascularization in the liver containing multiple preneoplastic nodules. These observations suggest that at an early stage of liver carcinogenesis reduction or loss of angiogenesis inhibitors may contribute to initiation of neoangiogenesis. A number of other identified proteins known to be associated with hepatomas are also present at early-stage neoplasia.

Endogenous transforming growth factor-beta receptor-mediated Smad signaling complexes analyzed by mass spectrometry.

ASmad proteins are the central feature of the transforming growth factor-beta (TGF-beta) intracellular signaling cascade. They function by carrying signals from the cell surface to the nucleus through the formation of a series of signaling complexes. Changes in Smad proteins and their complexes upon treatment with TGF-beta were studied in mink lung epithelial (Mv1Lu) cell cultures. A time course of incubation with TGF-beta was carried out to determine the peak of appearance of phosphorylated Smad2. Immobilized monoclonal antibody against Smad2 was then used to isolate the naturally occurring complexes. Three strategies were used to identify changes in proteins partnering with Smad2: separation by one-dimensional SDS-PAGE followed by MALDI peptide mass fingerprinting, cleavable ICAT labeling of the protein mixtures analyzed by LC-MS/MS, and nano-LC followed by MALDI MS TOF/TOF. Smad2 forms complexes with many other polypeptides both in the presence and absence of TGF-beta. Some of the classes of proteins identified include: transcription regulators, proteins of the cytoskeletal scaffold and other tethering proteins, motility proteins, proteins involved in transport between the cytoplasm and nucleus, and a group of membrane adaptor proteins. Although some of these have been reported in the literature, most have not been reported previously. This work expands the repertoire of proteins known to participate in the TGF-beta signal transduction processes.