In this study, we employed the 2DGE and MALDI-TOF/TOF-MS to identify the differentially expressed proteins in this cell line upon exposure to MNQ at 7.
Abbreviations: 2DGE, two-dimensional gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammoniol-1- propanesulfonate; AP-1, activator protein-1; DTT, dithiothreitol; DMSO, dimethyl sulphoxide; EMT, epithelial-to-mesenchymal transition; ERK1/2, extracellular signal-regulated kinase 1/2; FAR, focal adhesion kinase; FBS, fetal bovine serum; IPG, immobilised pH gradient; MALDI-TOF/TOF-MS, matrix-assisted laser desorption ionisation tandem time-of-flight mass spectrometry; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MNQ, 2-methoxy-1,4-naphthoquinone; NF-[kappa]B, nuclear factor-[kappa]B; NMIIA, nonmuscle myosin-IIA; ROS, reactive oxygen species; RPSA, laminin-binding protein; S100A4, protein S100-A4; uPa, urokinase plasminogen activator,
5] Nonstandard abbreviations: 2DGE
, 2-dimensional gel electrophoresis; ANIT, [alpha]-naphthylisocyanate; MS, mass spectrometry; MCI, master cluster index; MALDI, matrix-assisted laser desorption/ionization; ALT, alanine aminotransferase; PNP, purine nucleoside phosphorylase; AST, aspartate aminotransferase; MDH, malate dehydrogenase; MAT, methionine adenosyltransferase; and Apo, apolipoprotein.
ANALYSIS OF THE 2DGE PATTERN OF HUMAN PITUITARY PROTEOMES
High-resolution, 2DGE separation of proteins is a key step in performing a comparison between human pituitary proteomes.
Use of a commercial IPG strip (18-cm IPGstrip pH 3-10 NL) for the first-dimension IEF, a vertical multigel electrophoresis system (can analyze up to 12 gels at a time) for second-dimension SDS-PAGE, and an appropriate amount of protein loaded on the gel (~70 [micro]g) (13,14) allowed us to obtain a high-resolution, high-reproducibility 2DGE pattern of a human pituitary proteome.
The experimental pI values and molecular weights of each differential spot in Table 3 were obtained by inputting the standard 2DGE markers (Fig.
In this study, we found that most of the differential spots found across gender, age, and race were low-intensity 2DGE spots.
A high-resolution, high-reproducibility 2DGE map, which contains ~1000 proteins of a human pituitary proteome, was obtained.