An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Abstract

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.

Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle

“…Several earlier studies have collected proteomic data from the mammary tissue of lactating animals (1,6,14,16,18,48,50,60). None have studied mammary mitochondria.…”

“…1D). This assay demonstrated that mitochondrial ATP synthesis activity increased by twofold between day 2 and 8 postpartum, remained 1 The online version of this article contains supplemental material. elevated through day 14 postpartum, and then decreased on day 21 postpartum.…”

“…Details of the analysis including mean squares, the value of the F-statistic, and its corresponding P value are presented in Supplemental Table S3. 1 An overall ontology analysis was conducted on the list using Mouse Genome Informatics Database Gene Ontology Term Finder. Ingenuity Pathway Analysis (IPA; Ingenuity Systems, http://www.ingenuity.com) was used to conduct ontology on proteins within the dataset that exhibited statistically significant changes (P Ͻ 0.05).…”

Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis

“…Known modifications such as methionine and tryptophan oxidation, acetylation at the N-terminus, methylation, pyro-glutamylation and others may be checked, followed by searches with the findmod tool (http://us.expasy.org/tools/findmod/). This procedure complements the prediction of posttranslational modifications by iterative calibration of 2-DE gels [37]. In a third iteration the contaminant peaks should be reanalyzed to find out, whether they contain peptides of the main, neighbor spot, or additional components for further improvement of scoring factors and SC.…”

trans-11 18:1 Vaccenic Acid (TVA) Has a Direct Anti-Carcinogenic Effect on MCF-7 Human Mammary Adenocarcinoma Cells

“…Total cell proteins were obtained from cells grown in 100-mm dishes for 72 h after being treated [20,21]. Briefly, the cells were washed three times with ice-cold PBS after which they were lysed on ice in lysis buffer containing 8 M urea, 4% CHAPS, 5 mM EDTA, 10 mM Tris-HCl (pH 8.3), and protease inhibitors (GE Healthcare Corp., Piscataway, NJ, USA).…”