A two-dimensional protein map ofCaenorhabditis elegans

Abstract

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

Identification of proteins from two-dimensional polyacrylamide gels using a novel acid-labile surfactant

“…Mass spectrometry (MS), combined with separation methods such as PAGE and high-performance liquid chromatography (HPLC), has emerged as a key technology for studying the spatial and temporal expression of proteins [13]. Mass analysis is usually performed on peptides obtained by tryptic digestion of proteins following gel electrophoresis, LC or affinity purification [9,12,[13][14][15][16][17][18][19][20][21][22][23][24]. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) is a powerful method for peptide sequencing and protein identification, especially when combined with capillary electrophoresis or HPLC separation, automated MS/MS and database search procedures [13,18,21,[25][26][27][28].…”

“…MS identification of gel separated proteins requires compatible PAGE procedures [20]. Gels can be stained using colloidal Coomassie [3,14,16,20,22,30], zinc-imidazole [38,39], fluorescent [20,40] or modified silver stains [19,22,30,41,42] to visualize and target differentially expressed proteins for MS analysis. A surfactant is also required for solubilization, denaturing and transfer of focused proteins to the 2-D gel following IEF [37].…”