Figure 1. Schematic diagram of this protocol.

(i.) Cells will be grown in heavy and light SILAC media and will be transfected with a plasmid supporting (ii.) Inducible, ectopic L1 expression. From the moment the cells reach sufficient density to execute the transfection, (a.) it will be ~4 more days to harvest and freeze them; this is a natural stopping point in the protocol. These manipulations are described in Procedure A. (b.) An additional ~2 days will be needed to cryomill the cells to powder and carry out quality control checks by western blot and other protein assays, as well as to complete the peptide workup for mass spectrometry (MS), to determine the degree of heavy SILAC-label incorporation. The MS analysis could be completed as quickly as ~1 more day if done immediately. These manipulations are described in Procedure B. Pause until thorough SILAC labeling is confirmed. (iii.) Carry out affinity capture, followed by RNase and control treatments; these manipulations are described in Procedure C. (c.) Affinity capture and subsequent peptide workup (described in Procedure D) will require ~2 days. The time to complete (iv.) MS (Procedure D) and data analysis will depend on the number of samples but could be done within an additional ~3 days if the experiment is designed exactly as laid out in this protocol. Aside from the LC-MS/MS runs themselves, a substantial portion of the time is occupied by processing the RAW data files in MaxQuant; speed will vary according to the capabilities of the workstation.