Search tips
Search criteria 


Logo of jbtJBT IndexAssociation Homepage
J Biomol Tech. 2007 February; 18(1): 28–29.
PMCID: PMC2291982

P83-M Characterization of Pil1p and Lsp1p Phosphorylation in S. cerevisiae using Tandem Affinity Purification and LC-MS/MS


In order to elucidate the signaling pathway of the human mPR-α protein, a membrane progesterone receptor, a strain of S. cerevisiae overexpressing this protein was generated. Initial global screens using 2D gels, a phospho-specific stain, and tandem MS/MS identified 35 proteins exhibiting alterations in phosphorylation state in response to progesterone activation. Among these were Pil1p and Lsp1p, components of a newly discovered immobile protein mass, the eisosome, purported to denote endocytotic sites. However, due to their similar molecular weights, pI values, and a shared sequence identity of 72%, the separation achieved using 2D gel electrophoresis was insufficient to identify which of these two proteins was exhibiting a change in its phosphorylation state. The experiment presented here utilizes a strain of yeast containing an endogenously TAP (tandem affinity purification)-tagged version of Lsp1p. Since Pil1p and Lsp1p interact, the purification pulled out both of these eisosome proteins. Subsequent separation using 1D gels distinguished the two proteins due to the additional molecular mass imparted to Lsp1p by the tag. Both proteins were then excised, digested in-gel with trypsin, and analyzed via LC-MS/MS using a Q-Star XL mass spectrometer. A total of six unique phosphopeptides were identified: five in Pil1p and one in Lsp1p. The tag was then swapped onto Pil1p in order to identify any additional sites on Lsp1p that may have been suppressed due to the presence of the tag. The findings presented here represent a significant step towards our ultimate goal of monitoring changes in phosphoproteins in response to progesterone activation.

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities