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Two-dimensional gel electrophoresis (2DE) is a proven high-resolution separation technology in the protein laboratory. Nevertheless, ever since its invention the technique has had the drawback of low reproducibility. For protein expression analysis based on replicates, differential gel electrophoresis (DIGE) has, therefore, been invented. This technique is however not applicable for the comparison of singular samples. With comparative fluorescence 2DE (CoFGE) a method was developed, which creates a protein reference net for gel matching on top of the gel-separated protein analyte. This internal standard enables both the correction of the protein spot coordinates and the estimation of the analyte concentration. It extends the use of 2DE and truly enables the use of searchable, browser-based 2DE databases.

Amphoteric azo dyes were used for internal control of pI values in Comparative two-dimensional Fluorescence Gel Electrophoresis (CoFGE) [1]. The 2D-gel images of separated Escherichia coli proteins as well as those of colored amphoteric dyes separated by isoelectric focussing are presented. The latter were used to correct for variation in the first electrophoretic dimension and further improve protein coordinate assignment in 2D-gel electrophoresis. Data tables are supplied to demonstrate pI-value calibration and the effect on the assignment of protein spot coordinates.

Methyl mercaptan is derived from l -methionine by the action of l -methionine-α-deamino-γ-mercaptomethane lyase (METase) and is a major component of oral malodor. This compound is highly toxic and is thought to play an important role in periodontal disease. We found that Treponema denticola , a member of the subgingival biofilm at periodontal disease sites, produced a large amount of methyl mercaptan even at low concentration of l -methionine. METase activity in a cell-free extract from T. denticola was detected by two-dimensional electrophoresis under non-denaturing conditions, and the protein spot that exhibited high METase activity was identified using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The identified gene produced a METase with a K m value for l -methionine (0.55 mM) that is much lower than those of METases previously identified in the other organisms. This result suggests that T. denticola is an important producer of methyl mercaptan in the subgingival biofilm.

Purpose. The NOD mouse, as well as the newly constructed C57BL/6.NOD-Aec1Aec2 mouse, have been shown to exhibit numerous disease parameters that parallel Sjogren’s syndrome (SjS) in humans, including loss of stimulated fluid secretion concomitant with the appearance of leukocytic infiltrates in the lacrimal and salivary glands. In the present study, we have catalogued the temporal changes in tear proteins with the onset of dry eye disease in these two mouse strains. Methods. Tears, collected from CD-1, NOD.B10-H2b and C57BL/6.NOD-Aec1Aec2 mice at 3, 8, 14, and 20 weeks of age, were labeled with either Cy-3 (green) or Cy-5 (red) dye. Labeled tear proteins were separated individually on pH3-10 NL18 cm IPG strips, then electrophoresed through 8-16% Tris-HCl SDS-polyacrylamide gels. Each gel was scanned for visualization using ImageQuant software. Each gel was compared by ImageMaster 2-D Elite software to identify unique protein difference. Each protein spot was sequenced using MOLDI-TOF. Results. NOD.B10-H2b and C57BL/6.NODAec1Aec2 mice exhibited marked losses in their tear protein concentrations compared to CD-1 mice. To date, nearly 50 protein spots have been compared between the autoimmune and nonautoimmune mice. Some 10 tear proteins remained unchanged through 20 weeks of age (e.g., lacrimal androgen binding protein); however, the majority of tear proteins showed some increased levels in the autoimmune mice (e.g., DNAseI orglobulin IV precursor), while >12 spots were lost in conjunction with the onset of dry eye disease. Conclusions. Onset of dry eye disease in mice exhibiting SjS-like disease is associated with loss of tear volume, decreased tear protein concentrations, loss in expression of several tear proteins, plus appearance of altered proteins. Commercial Relationship(s): ABP is a scientific consultant for and stockholder in Ixion Biotechnology, Inc.; Support: NIH grants DE13769, DE014344 & DE015152

The abnormal gene product in cystic fibrosis has not been identified. We report that two-dimensional poly-acrylamide gel electrophoresis of 125 I-labeled sweat proteins coupled with fluorography and rare earth screen radioautography reveals an absence of an acidic protein of molecular weight 60,000 in sweat collected from 9 patients with cystic fibrosis. The protein spot was present in sweat from heterozygotes and from controls.