Immobilized pH gradient two‐dimensional gel electrophoresis and mass spectrometric identification of cytokine‐regulated proteins in ME‐180 cervical carcinoma cells

Abstract

Two-dimensional (2-D) polyacrylamide gel electrophoresis combined with mass spectrometry is a powerful combination of technologies that allows high resolution separation of proteins and their rapid identification. Immobilized pH gradient (IPG) first-dimensional gels have several advantages over carrier ampholyte isoelectric focusing, including a high degree of reproducibility, good protein spot resolution, and a selection of pH range. Here we demonstrate the utility and efficacy of combining IPG 2-D gel electrophoresis with mass spectrometry to identify interferon-gamma- (IFN) and tumor necrosis factor (TNF)-regulated proteins in ME-180 cervical carcinoma cells. Three cytokine-regulated proteins have been identified, using imidazole-zinc-stained preparative IPG 2-D gels and in-gel tryptic digestion followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for determination of peptide masses and sequences: 1) triosephosphate isomerase, a glycolytic pathway enzyme, 2) proteasome subunit C3, which is important in protein degradation, and 3) Ran, a GTP-binding protein important in cell cycle regulation, protein import into the nucleus, and RNA export from the nucleus.

Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus ( Luteoviridae )

“…By manually comparing the colloidal blue images with the 2D DIGE images, we were able to identify a set of spots that were unique to or obviously upregulated in vector or nonvector genotypes. These spots were then excised from the picking gels and digested with trypsin, and the resulting peptides were extracted for analysis by MS (19). Special attention was paid to unique protein spots associated with vector or nonvector phenotypes that were conserved in both parent aphids and F 2 offspring.…”

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

“…The introduction of imidazole for zinc staining overcame this problem in SDS-PAGE applications [101] and was further modified for application to non-SDS gels [102]. These marked improvements to zinc staining resulted in a large number of studies aiming to further enhance the method and demonstrate that proteins within zinc stained gels were still biologically active, could be recovered with high yields and were compatible with MS technologies [103][104][105][106][107][108][109][110]. Since this process leaves proteins presumably untouched, significant effort was invested in highlighting its qualitative potential and studies examining its quantitative capacity have been less prevalent.…”

“…The consensus for zinc-imidazole-stained gels Yes [20], [73], [157,158] 500 ng A. thaliana total protein SR, MeOH/HAc fixation, stain from 3 h to overnight, destain in MeOH/HAc, water washed. was also to take images, against a black background in this case, usually using a Polaroid camera or an automated imager, and follow with analysis [20,100,101,104,107,108,243]. Fluorescence posed a different challenge in terms of detection given the specific excitation and emission requirements of different fluorophores.…”

Peptidomics of a Single Identified Neuron Reveals Diversity of Multiple Neuropeptides with Convergent Actions on Cellular Excitability

“…To obtain peptide sequence information, post-source decay mass spectra were recorded on a VG Tofspec SE time-of-flight mass spectrometer (Micromass, Manchester, UK) equipped with a nitrogen laser operated in reflectron mode, as described previously (Matsui et al, 1997). Samples were prepared as described above.…”