Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry

Abstract

The nematode Caenorhabditis elegans (C. elegans) is the first animal whose whole 97 Mb genome sequence, encoding ca. 19000 open reading frames (ORF's), has been essentially determined. We tried to establish a 2-DE map of the nematode proteome by means of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A soluble protein fraction of mixed stages of the worm, wild-type strain N2, was applied to 2-D PAGE. After Coomassie Brilliant Blue (CBB) staining, 1200 spots were detected and 140 major spots were excised from the gel and subjected to in-gel digestion with Achromobacter protease I (lysyl endopeptidase). Resulting peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting for protein identification. With this approach we have obtained a two-dimensional electrophoresis (2-DE) protein map in which 69 spots were localized as landmarks for comparison of expression profiles to elucidate the basis of various biological events.

Genetic analysis of ETS genes in C. elegans

“…Protein expression pro®les from worms carrying gain or loss of function mutations in ETS genes could be produced by 2 dimensional (2D) electrophoresis. Dierentially expressed proteins can be excised directly from 2D gels and identi®ed by matrix assisted laser desorption/ionization-time of ight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass ®ngerprinting for protein identi®cation (Kaji et al, 2000). Alternatively, the complete mRNA expression pro®le of two populations of worms can be compared using DNA microarray technology.…”

Proteomic approaches to study epigallocatechin gallate-provoked apoptosis of TSGH-8301 human urinary bladder carcinoma cells: Roles of AKT and heat shock protein 27-modulated intrinsic apoptotic pathways

“…Protein spots were quantified using the ImageMaster 2D Elite software (GE Healthcare Biosciences) as previously described (49). The procedures were modified as described by Kaji et al (50). Protein spots excised from the CBB-stained gel were destained and dried completely.…”

An Overview of Toxicogenomics

“…Currently, the most commonly used technologies for proteomics research are 2-dimensional (2-D) gel electrophoresis for protein separation followed by mass spectrometry analysis of proteins of interest (Rasmussen, et al, 1994;Shaw, et al, 1999;Carroll, et al, 2000;Fountoulakis, et al, 2000;Kaji, et al, 2000;Watarai, et al, 2000). Analytical protein characterization with multidimensional liquid chromatography/mass spectrometry improves the throughput and reliability of peptide identity.…”