Hippocampal levels of gamma-enolase, C-1-tetrahydrofolate synthase and serotransferrin fluctuate over the estrous cycle in the rat.

Hippocampal functions vary across the estrous cycle but metabolic changes at the protein level have not been systematically studied so far. It was therefore the aim of the study to screen expression of metabolic proteins mainly represented by metabolic enzymes in the hippocampus over the estrous cycle and in males. Female and male OFA Sprague-Dawley rats were used and female estrous phases were determined by vaginal smears, according to which females were separated into groups of proestrous, estrous, early and late metestrous and diestrous. Proteins were extracted from hippocampal tissue and separated on two-dimensional gel electrophoresis followed by identification with mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Comparative analysis of protein levels was carried out by quantifying protein spot volumes by means of specific software. Levels of one expression form of gamma-enolase were different between diestrous and early metestrous; C-1-tetrahydrofolate synthase levels were elevated in proestrous as compared with estrous and serotransferrin levels were increased in diestrous as compared with proestrous, estrous, metestrous and in males. The outcome of estrous cycle- and gender-dependent protein fluctuations is relevant for the interpretation of previous and future work as well as for the design of further studies at the protein level in the hippocampus.

Mass spectrometrical analysis of the processed metastasis-inducing anterior gradient protein 2 homolog reveals 100% sequence coverage.

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia. Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF. The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed. Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.

Expression of proteasomal proteins in ten different tumor cell lines.

Controlled intracellular protein degradation is crucial for the maintenance of normal cell functions. An evolving concept claims that alterations in the exact timely degradation of proteins involved in growth control, apoptosis, signaling and differentiation contribute to carcinogenesis. This tightly regulated process is facilitated by the ubiquitin-26S proteasome system, a multi-enzyme complex, and inhibitors of this pathway have already been developed as potential anticancer agents. In order to generate proteasomal protein expression patterns of tumor cells and to provide an analytical tool we applied two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MALDI-TOF-TOF with LIFT technology) in ten individual tumor cell lines (Saos-2; SK-N-SH; HCT-116; Caov3; A-549; HL60; A-673; A-375; MCF-7; HeLa) widely used in tumor research. A series of 39 proteasomal/proteolytic proteins was unambiguously identified by this proteomic approach, comprising proteins of the 20S core complex, the 19S regulatory complex, the 11S regulator, components of the ubiquitin pathway and proteases. Construction of individual protein maps by 2-DE and mass spectrometry provides an analytical tool and reference base for studying the pivotal importance of the proteasome and other proteolytic enzymes in tumor cells, independent of antibody availability and specificity. This preliminary database enables for designing studies in this area of research and reveals proteins that can be used as targets for new therapeutic strategies.