Phospho-proteomic approach to identify new targets of leucine deprivation in muscle cells.
The aim of this study was to optimize a protocol that allows identifying changes at the phosphorylation level of specific proteins in response to cell stimulation by leucine starvation. To make possible the identification of differentially phosphorylated proteins by the combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), we prepared fraction enriched in phosphoproteins. For that purpose, we adapted the immobilized metal affinity chromatography (IMAC) technique to make it compatible with 2D-PAGE. On the whole, this procedure allowed identifying regulated targets of leucine deprivation: molecular chaperones glucose-regulated protein 58 kDa (GRP58) and BiP (GRP78), RNA helicase DEAD box polypeptide 3, and eukaryotic translation initiation factor 4B (eIF4B).
Talvas
Jérémie
J
Unité de Nutrition Humaine, INRA, Centre de Clermont-Ferrand/Theix, F-63122 Saint-Genès-Champanelle, France.
Obled
Alain
A
Sayd
Thierry
T
Chambon
Christophe
C
Mordier
Sylvie
S
Fafournoux
Pierre
P
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D007930:Leucine / Q000172:deficiency*; D051379:Mice; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism*; D032446:Myoblasts / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis*; D040901:Proteomics; D015203:Reproducibility of Results; D020123:Sirolimus / Q000494:pharmacology
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717248:Hspa5 protein, mouse; D010750:Phosphoproteins; D007930:Leucine; D020123:Sirolimus