MASS-SPECTROMETRIC ANALYSIS OF 40-S RIBOSOMAL-PROTEINS FROM RAT-1 FIBROBLASTS

Although sequences of most mammalian ribosomal proteins are available, little is known about the posttranslational processing of ribosomal p roteins. To exam ine their post-translational modifications, 40 S subu nit proteins purified from Rat-1 fibroblasts and their peptides were a nalyzed by Liquid chromatography coupled with electrospray mass spectr ometry. Of 41 proteins observed, 36 corresponded to the 32 rat 40 S ri bosomal proteins with known sequences (S3, S5, S7, and S24 presented i n two forms). The observed masses of S4, S6-S8, S13, S15a, S16, S17, S 19, S27a, S29, and S30 matched those predicted. Sa, S3a, S5, S11, S15, S18, S20, S21, S24, S26-S28, and an 57 variant showed changes in mass that were consistent with N-terminal demethionylation and/or acetylat ion (S5 and S27 also appeared to be internally formylated and acetylat ed, respectively). S23 appeared to be internally hydroxylated or methy lated. S2, S3, S9, S10, S12, S14, and S25 showed changes in mass incon sistent with known covalent modifications (+220, -75, +86, +56, -100, -117, and -103 Da, respectively), possibly representing novel post-tra nslational modifications or allelic sequence variation. Five unidentif ied proteins (12,084, 13,706, 13,741, 13,884, and 34,987 Da) were obse rved; for one, a sequence tag (PPGPPP), absent in any known ribosomal proteins, was determined, suggesting that it is a previously undescrib ed ribosome-associated protein. This study establishes a powerful meth od to rapidly analyze protein components of large biological complexes and their covalent modifications.