title: Two-dimensional gel electrophoresis of gastric tissue in an alkaline pH range.
abstract: 2DE in combination with MS has facilitated the discovery of several proteins with altered abundance in gastric cancer. While acidic and wide pH ranges have been widely investigated, analysis in the alkaline pH range has not been specifically performed in gastric cancer to date. In the present study, we initially optimized the 2DE in alkaline pH range (pH 7-11) for gastric tissue samples. Using a modified lysis buffer, we analyzed pooled nontumor and tumor samples for proteins with altered abundance in gastric adenocarcinoma. We successfully identified 38 silver-stained spots as 24 different proteins. Four of these were chosen for investigation with immunoblotting on individual paired samples to determine whether the changes seen in 2DE represent the overall abundance of the protein or possibly only a single form. While mitochondrial trifunctional protein (MTP) subunits were decreased in 2DE gels, immunoblotting identified their overall abundance as being differently dysregulated: in the gastric tumor samples, the MTP-α subunit was decreased, and the MTP-β subunit was increased. On the other hand, heterogenous nuclear ribonucleoprotein M and galectin-4 were increased in the gastric tumor samples in both 2DE and immunoblotting.
authors.affiliation: Medical Centre for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Nina
authors.initials: N
authors.lastname: Kočevar
authors.forename: Snježana Frković
authors.initials: SF
authors.lastname: Grazio
authors.forename: Radovan
authors.initials: R
authors.lastname: Komel
mesh_terms: D000230:Adenocarcinoma / Q000737:chemistry* / Q000473:pathology; D014408:Biomarkers, Tumor / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing / Q000379:methods; D008297:Male; D020543:Proteome / Q000032:analysis*; D013270:Stomach / Q000737:chemistry / Q000473:pathology*; D013274:Stomach Neoplasms / Q000737:chemistry* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; D020543:Proteome
keywords: 2DE; Biomedicine; Gastrointestinal cancer; Isoelectric focusing; Protein biomarker discovery; pH