A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

Abstract

Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1), relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D) electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IkappaB kinase b (IkappaBKb) and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

Figure 1

Batch Ion-Exchange of soluble C2C12 proteins increases the total number of spots visualized 2.5-fold. Serum-starved C2C12 cells were stimulated for 2 hours with 100 nM IGF-1 and lysed in detergent-free buffer to generate soluble proteins. 100 ug of protein was separated by 2D electrophoresis followed by silver staining (left). Another 100 ug of protein was further fractionated by batch ion-exchange followed by desalting/concentration [17] before 2D electrophoresis and silver staining. The total amount of protein detected was analyzed using PDQuest software (Bio-Rad).

Figure 2

IGF-1 treatment of C2C12 cells activates MAP kinase and PI 3-kinase signaling pathways. C2C12 cells were serum-starved for 18 hours prior to stimulation with IGF-1 (100 nM) for 0, 1, 2, 4, 8, or 24 hours. Cells were lysed in detergent containing buffer and 20 ug of protein from each time point was separated on a 10% SDS polyacrylamide gel. Western blots were performed using phosphospecific antibodies to Erk (p42/p44) and Akt (p473) as well as antibodies that detect total protein. Actin is shown as a loading control.

Figure 3

Analysis of protein expression changes during IGF-1 stimulation. A) Visualization of significant and reproducible changes in protein expression in IGF-treated C2C12 analyzed as above. (Top panel) Expression of proteins highlighted by a yellow circles increase after an 8 hour treatment with IGF-1. (Bottom panel) Proteins with red circles are ones that disappear after 8 hour IGF-1 treatment, while proteins within the blue arrows shift position in response to IGF-1 stimulation (ie, possible post-translational modifications including phosphorylation, acetylation, methylation).

Figure 4

Expression of Rho-GDI, Cofilin, PDGF receptor α, and MAPKBP-1 decreases upon IGF-1 stimulation of C2C12 cells. A) Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain. The protein highlighted by a yellow square (Rho-GDI, Cofilin, PDGF receptor α, and MAPKBP-1) decrease in expression after IGF-1 treatment. PDQuest analysis indicates a quantitative change in the protein based on total quantitation of the entire gel. B) Western blot analysis confirms the sustained decrease in protein expression for Rho-GDI, cofilin, and the PDGF receptor.

Figure 5

Expression of Rad50, enolase, IκB kinase b, and Hsp70 increases upon IGF-1 stimulation of C2C12 cells. A) Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain. The protein highlighted by a yellow square (Rad50, enolase, IκB kinase b, and Hsp70) increase in expression after IGF-1 treatment. PDQuest analysis indicates a quantitative change in the protein based on total quantitation of the entire gel. B) Western blot analysis confirms the sustained increase in protein expression for Rad50, enolase, IκB kinase b, and Hsp70. C) RAD50 accumulates in the cytosolic fraction of IGF-1 stimulated C2C12 cells.

Figure 6

Triplicate samples from three different C2C12 cell preparations were stimulated with IGF-1 (100 ng/ml) for up to 8 hours, fractionated using batch ion-exchange, separated by 2D electrophoresis, and proteins were detected with silver stain. The protein highlighted by a yellow square (eIF-4H) rapidly increases in expression after 1 hour IGF-1 treatment, but then decreases at 4 and 8 hours.