A density-based proteomics sample fractionation technology: folate deficiency induced oxidative stress response in liver and brain

Abstract

Folate deficiency (FD) alters hepatic methionine metabolism and is associated with increased hepatocellular apoptosis. Additionally, mice deprived of folate showed increased oxidative damage in brain tissue, leading to cognitive impairment. Most previous studies have focused independently on either liver, the main tissue of folate storage and metabolism, or brain, where folate regulates neurogenesis and programs cell death. The aim of this study was to apply a powerful, rapid proteomics approach to understand potential subcellular correlations of folate deficiency in both brain and liver of the same rat. This approach combined a new density-based sample fractionation technology (enhanced density gradient extraction = Edge technology) with other conventional proteomics techniques, such as western blot analysis, 2DE, and mass spectrometry. The brain and the liver from individual rats, fed normal or FD diets for 6 wks, were homogenized and then fractionated using the Edge 200 Separation System. Subsequently, all fractions from brain and liver, from control and treated rats, were analyzed by western blot using two markers of oxidative stress: glutathione peroxidase 1 (GPx1) and glucose-regulated protein 75 (GRP75). certain fractions were selected based on western blot analysis and were further analyzed by 2DE. protein spots of interest were identified by MALDI-TOF/TOF. The results demonstrated that edge technology provides a powerful density based separation and enrichment method for rapid screening of potential FD markers and their possible correlations to both liver and brain diseases.

FIGURE 1

PNS of rat livers and brains was fractionated using the Edge 200 Separation System. The step-wise density gradient extraction process is described as follows: 3 ml of PNS in 250 mm (~8.5%) sucrose was loaded in a rotor sample container; the rotor sample container in a rotor was spun for 30 min to pellet all the subcellular particles except soluble proteins and very small particles. The supernatant was removed. The pellet was resuspended in 10% sucrose solution and spun for 2 min. After the supernatant was removed, the pellet was resuspended in 15% sucrose solution and spun for 2 min. The process was continued in the same manner through the last fraction, which was 60% sucrose. The speed of the rotor was 95K rpm with an average 120K g force.

FIGURE 2

Edge 200 Separation System with sample rotor and rotor sample container.

FIGURE 3

Western blot analysis of rat liver PNS from control and FD rats before fractionation. Equal amount of total protein was subjected to western analysis as described under Materials and Methods. A: Relative expressions of Gpx1 of liver pns in three control and three FD rats. B: Relative expressions of GRp75 of liver pns in three control and three FD rats.

FIGURE 4

Western blot analysis of rat brain PNS from control and FD rats before fractionation. Equal amount of total protein was subjected to western analysis as described under Materials and Methods. A: Relative expressions of Gpx1 of brain pns in three control and three FD rats. B: Relative expressions of GRp75 of brain pns in three control and three FD rats.

FIGURE 5

Western blot analysis of GPX1 of fractionated liver from control and FD rats. Relative percentage distribution of each fraction was calculated from the intensity of immuno-signal from each fraction divided by the sum of intensity of all fractions. The western blot images from rat #3 (ctrl) and rat #6 (FD) are shown.

FIGURE 6

Western blot analysis of GRP75 of fractionated liver from control and FD rats. Relative percentage distribution of each fraction was calculated from the intensity of immuno-signal from each fraction divided by the sum of intensity of all fractions. The western blot images from rat #3 (ctrl) and rat #6 (FD) are shown.

FIGURE 7

Western blot analysis of GPX1 of fractionated brain from control and FD rats. Relative percentage distribution of each fraction was calculated from the intensity of immuno-signal from each fraction divided by the sum of intensity of all fractions. The western blot images from rat #2 (ctrl) and rat #5 (FD) are shown.

FIGURE 8

Western blot analysis of GRP75 of fractionated brain from control and FD rats. Relative percentage distribution of each fraction was calculated from the intensity of immuno-signal from each fraction divided by the sum of intensity of all fractions. The western blot images from rat #2 (ctrl) and rat #5 (FD) are shown.

FIGURE 9

Details of 2D gel images of liver fractions 30% and 40%, showing selected differentially regulated proteins. Fractions from control rat #3 and FD rat #6 were selected for 2D gel analysis as described under materials and methods.

FIGURE 10

MALDI-TOF/TOF spectra of protein “similar to similar to 6-phosphobluconate dehydrogenase, decarboxylating,” identified from 2D gel spot number 8507 of rat liver 40% sucrose fraction. A: Total ion scan. B: ms/ms spectrum of the precursor 1591.81. C: ms/ms spectrum of the precursor 2160.12.

FIGURE 11

MALDI-TOF/TOF spectra of protein “similar to alpha-1 major acute phase protein prepeptide,” identified from 2D gel spot number 3607 of rat liver 30% sucrose fraction. A: Total ion scan. B: ms/ms spectrum of the precursor 1295.71. C: ms/ms spectrum of the precursor 1676.79.

FIGURE 12

2D gel images overlay of control and FD rat brain fraction 40%. The overlay image was performed by the PDQest software. Control rat #3 and FD rat #6 were selected for 2D gel analysis, as described under Materials and Methods.