Figure 2: Identification and validation of two novel peptides at the FARP1 locus.

(a) Screenshot from the UCSC genome browser showing total RNA, ribosomal footprints, Refseq gene structure and degree of conservation (Phast Cons). The bottom part is a zoomed in version spanning intron 13. The arrows indicate the position of the two novel peptides. The first peptide (DGIRPSNPQPPQPSTGPASR) spans an exon–intron boundary; the underlined amino acids are located within the intron. The figure also indicates peptides found from ribosomal footprinting studies. (b) Average read density (RPKM) for all FARP1 introns. (c) RT–PCR gel showing RNA expression of three regions within FARP1 intron from depolarized cortical cells. (d) WB analysis of mouse brain lysate showing FARP1 protein bands of different molecular weights. Coomassie stained SDS–PAGE gel showing IP of FARP1 from mouse brain lysate. The bands were analysed using LC–MS/MS and confirmed as FARP1 (F8VPU2) protein along with peptides from intron (the full gel images of c,d are in Supplementary Fig. 11). (e) Chemical validation of one of the novel peptides (HSSLIDDMFR) from FARP1 by comparing its spectra with synthetic peptide spectra as described in Methods. The identified peptide (HSSLIDDMFR) spectra (top) and synthetic peptide spectra (bottom) show a strong match with a spearman correlation coefficient (ρ) of 0.51 and a significant P value of 1.4e−04. The charge state, m/z value, P value and rho value of the peptide are listed. Overlapped fragmentation table indicating the y and b ions that were detected in both ‘identified’ and ‘synthetic’ spectra. The green ions were detected in both spectra, blue were detected only in ‘identified’ spectra. (f) A model showing the domains’, disordered regions’ and regulatory regions’ prediction in FARP1 amino-acid sequence, including the novel coding region of intron 13 by ELM ( http://elm.eu.org/). The total number of regulatory regions per 100 amino acids is shown by line graph, out of which the distribution of a subset of functionally important regions per 100 amino acids are indicated by color bars. The number of regulatory regions is enriched in intron 13 coding region.