Figure 1: Overview of the experimental procedure and peptide identification.

(a) Workflow of the KCl depolarization experiment illustrating the collection of total RNA and protein from mouse cortical neurons. Protein samples from multiple time points after depolarization were digested, labelled with isobaric tags and analysed by quantitative proteomic analyses (LC–MS). (b) The resulting spectra were subjected to the database search procedure for filtering and matching peptide spectra. Twenty percent of the spectra matched to the known mouse proteome (Uniprot 12 December 2011, incl. canonical and isoform sequences). The remaining 904,922 spectra were searched against the custom RNA-Seq transcriptome database in six frames and the resulting matched spectra were filtered using stringent criteria as mentioned in the text and of these 250 novel peptides were selected for further analyses. The custom transcriptome was derived from the same samples described.